journal of blood group serology and molecular genetics · cd59, and augustine blood group systems....

Journal of Blood Group Serology and Molecular Genetics

Vo lu m e 34, Nu m b e r 3 , 2018

This issue of Immunohematology is supported by a contribution from

Grifols Diagnostics Solutions, Inc.

Dedicated to advancement and education in molecular and serologic immunohematology

85 Re v i e w

Proceedings from the International Society of Blood Transfusion Working Party on Immunohaematology, Workshop on the Clinical Significance of Red Blood Cell Alloantibodies, September 2, 2016, Dubai

Clinical significance of antibodies to antigens in the Raph, John Milton Hagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, and Augustine blood group systemsM. Moghaddam and A.A. Naghi

103 Re v i e w

Proceedings from the International Society of Blood Transfusion Working Party on Immunohaematology, Workshop on the Clinical Significance of Red Blood Cell Alloantibodies, Friday, September 2, 2016, Dubai

Clinical significance of antibodies to antigens in the Scianna, Dombrock, Colton, Landsteiner-Weiner, Chido/Rodgers, H, Kx, Cromer, Gerbich, Knops, Indian, and Ok blood group systemsS. Lejon Crottet

91 SeRo lo g i c Me t h o d Re v i e w

Rouleaux and saline replacementK.L. Waider


Utility of chloroquine diphosphate in the blood bank laboratoryT. Aye and P.A. Arndt


Detecting polyagglutinable red blood cellsC. Melland and C. Hintz

93 oRi g i n al Rep o Rt

Method-specific and unexplained reactivity in automated solid-phase testing and their association with specific antibodiesM.E. Harach, J.M. Gould, R.P. Brown, T. Sanders and J.H. Herman

109 caSe Rep o Rt

A delayed and acute hemolytic transfusion reaction mediated by an anti-c in a patient with variant RH allelesT.K. Walters and T. Lightfoot

118 in MeM o Ri a M

Marjory Stroup WaltersJ. Hegarty and T.S. Casina

ImmunohematologyJournal of Blood Group Serology and Molecular Genetics

Volume 34, Number 3, 2018

CONTENTS

120an n ou n ceM en tS

127adv eRt iSeM en tS

131inSt Ruct i o nS fo R au t h o RS

134Su bScRi p t i o n in fo RM at i o n

ii IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018

Immunohematology is published quarterly (March, June, September, and December) by the American Red Cross, National Headquarters, Washington, DC 20006.

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Copyright 2018 by The American National Red Cross ISSN 0894-203X

ed i to R- i n-ch i ef

Sandra Nance, MS, MT(ASCP)SBBPhiladelphia, Pennsylvania

Ma n ag i n g ed i to R

Cynthia Flickinger, MT(ASCP)SBBWilmington, Delaware

tec h n i ca l ed i to RS

Janis R. Hamilton, MS, MT(ASCP)SBBDetroit, Michigan

Christine Lomas-Francis, MScNew York City, New York

Joyce Poole, FIBMSBristol, United Kingdom

Dawn M. Rumsey, ART(CSMLT)Norcross, Georgia

Tiffany Walters, MT(ASCP)SBBCM

Charlotte, North Carolina

Sen i o R Med i ca l ed i to R

David Moolten, MDPhiladelphia, Pennsylvania

aS S o c i at e Med i ca l ed i to RS

P. Dayand Borge, MDPhiladelphia, Pennsylvania

Corinne L. Goldberg, MDDurham, North Carolina

Mo lec u l a R ed i to R

Margaret A. Keller, PhDPhiladelphia, Pennsylvania

ed i to R i a l aS S iSta n t

Linda Frazier

pRo d u ct i o n aS S iSta n t

Linda Frazier

co p y ed i to R

Frederique Courard-Houri

pRo o f R e a d eR

Wendy Martin-Shuma

elect Ro n i c pu b l i S h eR

Paul Duquette

ed i to R i a l boa R d

Patricia Arndt, MT(ASCP)SBBPomona, California

Barbara J. Bryant, MDGalveston, Texas

Lilian M. Castilho, PhDCampinas, Brazil

Martha R. Combs, MT(ASCP)SBBDurham, North Carolina

Geoffrey Daniels, PhDBristol, United Kingdom

Anne F. Eder, MDWashington, District of Columbia

Melissa R. George, DO, FCAPHershey, Pennsylvania

Julie K. Karp, MDPhiladelphia, Pennsylvania

Jose Lima, MDDouglassville, Georgia

Christine Lomas-Francis, MScNew York City, New York

Geralyn M. Meny, MDSan Antonio, Texas

Paul M. Ness, MDBaltimore, Maryland

Thierry Peyrard, PharmD, PhDParis, France

S. Gerald Sandler, MDWashington, District of Columbia

Ira A. Shulman, MDLos Angeles, California

Jill R. Storry, PhD Lund, Sweden

Nicole ThorntonBristol, United Kingdom

eM eR i t uS ed i to RS

Delores Mallory, MT(ASCP)SBBSupply, North Carolina

Marion E. Reid, PhD, FIBMSBristol, United Kingdom

on ou R cov eR

Perhaps Gustav Klimt’s best-known work, The Kiss (1907), dazzlingly melds the sensual with the abstract. The painting depicts a man and woman intertwined, he standing, bowed, while she kneels on an idealized quilt-like meadow of flowers. Their proximity to the top of the painting heightens the sense of intimacy and also suggests the possibility of transcending worldly constraints. We see little of the lovers—the back of his head, her face, their hands and feet, simply rendered but wrapped in golden raiment decorated with distinct mosaics. The man’s figure is comprised of juxtaposed rectangles and the woman’s of clustered circles, a geometry that hints at both the contrast and complement of their union. Despite its shapelessness, the gilded mass of clothing serves to intensify and exalt the physical act of the kiss and thus consecrates the couple and love itself. This issue includes Waider’s review on the use of saline replacement to identify rouleaux—the stacked coin appearance of the red cells likened to “clustered circles.”

David Moolten, MD

IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018 85


Clinical significance of antibodies to antigens in the Raph, John Milton Hagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, and Augustine blood group systemsM. Moghaddam and A.A. Naghi

Revie w

This article reviews information on the clinical significance of antibodies to antigens in the Raph, John Milton Hagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, and Augustine blood group systems. Antibodies to many of the antigens in these groups are rarely encountered because of the high prevalence of the associated antigens in most populations. For many of these antibodies, the clinical significance—that is, the potential to cause reduced survival of transfused antigen-positive red blood cells or a transfusion reaction (e.g., anti-P, anti-Jra, and anti-Lan), and/or hemolytic disease of the fetus and newborn (e.g., anti-RHAG4 and anti-Vel)—has been documented. For other antibodies, their prevalence is so rare that information on the clinical significance of their antibodies is not available (e.g., anti-FORS1). Immunohematology 2018;34:85–90.

Key Words: clinical significance, antibodies to red blood cell antigens, Raph, John Milton Hagen, I, Globoside, Gill, Rh-associated glycoprotein, FORS, JR, LAN, Vel, CD59, Augustine

Raph Blood Group System

The Raph blood group system contains just one antigen, MER2 (RAPH1), located on the tetraspanin CD151 glycoprotein (TM4SF).1,2 The true MER2– phenotype, associated with the presence of anti-MER2, is very rare and results from mutations in CD151, but there is a quantitative red blood cell (RBC) polymorphism in which RBCs of about 8 percent of white individuals are serologically MER2–.2

Clinical SignificanceThere have been six reports of human alloantibodies to

MER2. Three of the subjects were found to have a stop codon in the CD151 gene, which encodes a member of the tetraspanin family of proteins. These three individuals had nephropathy and deafness, and two of the three, who were siblings, also had skin lesions, deafness, and β-thalassemia minor. The fourth subject had missense mutation c.533G>A (p.Argl78His). Subjects 5

and 6 shared missense mutation c.511C>T (p.Argl71Cys) as well as a synonymous single-nucleotide mutation (c.579A>G) and had no clinical features. Although the CD151 protein is critical to cell adhesion and signaling and is implicated in cancer progression, its significance in transfusion medicine is limited to only one report of a hemolytic transfusion reaction (HTR).3 Least-incompatible RBC units should be selected for transfusion to patients with anti-MER2.2 No information on anti-MER2 causing hemolytic disease of the fetus and newborn (HDFN) is available.4

John Milton Hagen Blood Group System

The John Milton Hagen (JMH) blood group system consists of six high-prevalence antigens that are recognized by the International Society of Blood Transfusion (ISBT) and are numbered sequentially from JMH1 through JMH6. Confirmed JMH variants are named with the first letter from the antibody maker’s first name following JMH (JMH2 named JMHK, JMH3 named JMHL, JMH4 named JMHG, JMH5 named JMHM, JMH6 named JMHQ).5 These antigens are located on the Sema7A protein.5,6 The chromosomal location of SEMA7A is 15q22.3-q23. JMH1 is the primary antigen in the system and is present in greater than 99 percent of all individuals. The JMH1– phenotype is more commonly acquired by depression of the antigen. This finding might explain the serologic observation of a positive direct antiglobulin test (DAT) seen in many individuals with anti-JMH.5

Clinical SignificanceJMH1, commonly known as JMH, is most notable because

transient depression of the antigen occurs, and (auto)anti-JMH may develop.5 JMH– patients with anti-JMH often have no history of transfusion or pregnancy. Of seven JMH antibodies,

86 IMMUNOHEMATOLOGY, Volume 34, Number 3, 2018

five were IgG4 and two were IgG1, although IgG3 anti-JMH has been described. There are numerous cases where patients with anti-JMH have been transfused with JMH+ blood with no adverse effects. One such patient received 20 units of JMH+ blood in 10 months, with the expected hemoglobin rise. There are no reports of JMH antibodies causing HDFN, which is unsurprising considering that JMH antigens are expressed very weakly on cord cells. One patient with anti-JMH is reported to have experienced an acute intravascular HTR, but evidence that the JMH antibody was responsible is limited. Antibodies developed in the rare JMH variant types may cause reduced RBC survival.2 Today, rapid detection of JMH antibodies with recombinant SEMA7A protein and the particle gel immunoassay has been developed.7

I Blood Group System

The I antigen, together with i, used to be part of the Ii blood group collection. The gene encoding the I β-1, 6-N-acetylglucosamine transferase (IGNT/GCNT2) respon-sible for converting i active straight chains of carbohydrates to I active branched chains has been cloned,8 and some mutations responsible for adult i phenotype have been identified.9,10 Hence, I has been promoted to the I blood group system comprising only a single antigen, the I antigen, and i remains in the Ii collection. RBCs from adults predominantly express I antigens and only low levels of i antigens, higher levels of the latter predominate in fetal and neonatal RBCs. In a small number of individuals, only very low levels of I can be detected, and their RBCs show high levels of i (adult i phenotype). This phenotype is believed to result from lack of activity of the I branching transferase, a product of the GCNT2 gene.2

Clinical SignificancePotent cold reactive antibodies responsible for cold

agglutinin disease are usually of I specificity. These antibodies are generally monoclonal and are usually IgM, but IgG autoanti-I can also occur. They directly agglutinate I+ RBCs at 4°C with varying thermal amplitude but are generally inactive above 30°C. One autoanti-I active at 30°C caused an acute HTR in a small child when 2 units of blood were transfused immediately after removal from the refrigerator. Transient, polyclonal, or oligoclonal autoanti-I may arise from infection, most typically by Mycoplasma pneumoniae.

Alloanti-I of high titer is rare and usually presents in the sera of i adults. These antibodies are almost invariably IgM and are active only at low temperatures. Rare examples may

be hemolytic and have a thermal range up to 37°C, and some anti-I with a thermal range below 37°C can cause shortened survival of transfused I+ RBCs. One anti-I became potentially clinically significant after transfusion of 6 units of I+ blood.

Globoside Blood Group System

The P blood group antigen of the Globoside system is a glycolipid structure, also known as globoside, on the RBCs of almost all individuals worldwide. The P antigen (Gb4) is intimately related to the Pk and NOR (P1PK4) antigens.

The molecular genetic basis of globoside deficiency is the absence of functional P synthase caused by mutations at the B3GALNT1 locus. Other related glycolipid structures, the LKE and PX2 antigens, remain in the Globoside blood group collection pending further evidence concerning the genes and gene products responsible for their synthesis.11

Clinical SignificanceAnti-P is found in the serum of all Pk individuals and

can be separated from serum of p individuals by adsorption with P1

k or P2k cells or by inhibition with hydatid cyst fluid.2

When complement is present, anti-P will hemolyze P1 or P2 phenotype RBCs.2 P antibodies are IgM and often also IgG, are usually reactive at 37°C, and can cause severe intravascular HTRs. Autoanti-P is associated with paroxysmal cold hemoglobinuria. P antigen is also a receptor of parvovirus B19.

Cytotoxic IgM and IgG3 antibodies directed against P or Pk antigens are associated with a higher-than-normal rate of spontaneous abortion in women with the rare p, P1

k, and P2k

phenotypes.4

Gill Blood Group System

The Gill blood group system was added to the list of systems already recognized by the ISBT in 2002. GIL, the only antigen of the Gill system, is an antigen of high prevalence located on the water and glycerol channel aquaporin-3 (AQP3).2,12 The GIL– phenotype results from homozygosity for a splice mutation in AQP3. Anti-GIL has been identified in five Gil– white women who had been pregnant at least twice.2

Clinical SignificanceFive examples of anti-GIL have been identified, all in

white women who had been pregnant at least twice. No GIL– individual was found by screening 23,251 white American and 2841 African American women with anti-GIL. RBCs from two

M. Moghaddam and A.A. Naghi


ISBT conference—clinical significance of antibodies

of the babies of mothers with anti-GIL gave a positive DAT, but there were no clinical symptoms of HDFN. Anti-GIL may have been responsible for an HTR, and results of monocyte monolayer assay (MMA) with two GIL antibodies suggested a potential to cause accelerated destruction of transfused GIL+ RBCs.2

The Rh-Associated Glycoprotein Blood Group System

In 2010, the recognition that three RBC surface antigens were located on RhAG encoded by RHAG led to the establishment of a new blood group system. Two high-prevalence antigens, Duclos (RHAG1) and DSLK (RHAG3), two low-prevalence antigens, OI(a) and (RHAG2), and RHAG4, have serologic characteristics suggestive of expression on RhAG, but RHAG4 has been shown to not exist and is under investigation by ISBT to be retracted.13

Clinical SignificanceNo data are available.4

FORS Blood Group System

This blood group system has been named FORS after its original finder, Lund professor John Forssman. The FORS antigen (originally recognized as the Apae phenotype) was discovered by weak reactivity of RBCs against polyclonal anti-A reagents, reactivity against the lectin Helix pomatia (snail anti-A), and no reactivity with the plant anti-A1 lectin, Dolichos biflorus, in two different families. Genomic analysis of the ABO locus in both samples revealed that they were genetically group O, and the reactivity described must be due to other phenomena.14,15

Clinical SignificanceThe clinical significance of anti-FORS1 is not known.4

JR Blood Group System

The JR blood group system (ISBT 032) consists of one antigen, Jra, which is highly prevalent in all populations (>99%). Jra is located on the ABCG2 transporter, a multipass membrane glycoprotein (also known as the breast cancer resistance protein [BCRP]), which is encoded by the ABCG2 gene on chromosome 4q22.1.16–19 The rare Jr(a–) phenotype has been found mostly in Japanese and other Asian populations, but also in people

of northern European ancestry, in Bedouin Arabs, and in one Mexican individual. The rare Jr(a–) phenotype mostly results from recessive inheritance of ABCG2 null alleles caused by frameshift or nonsense changes.17,20–22 To date, more than 25 different mutations responsible for the absence of ABCG2 as well as mutations giving rise to weakened Jra expression have been identified.16

Clinical SignificanceABCG2 expression levels in cord RBCs are higher than

those in adult RBCs, and the change of ABCG2 expression in erythroid lineage cells may influence the clinical course of fetal anemia with anti-Jra.23 Anti-Jra may be stimulated by transfusion or pregnancy and has been detected in untransfused Jr(a–) women during their first pregnancy.24 Most anti-Jra are IgG1 and sometimes IgG3. Anti-Jra may fix complement, can be a dangerous antibody in pregnancy, and has been implicated in severe and fatal HDFN; in other pregnancies with anti-Jra, however, indications of HDFN have been no more than a positive DAT on cord cells or mild neonatal jaundice. Many transfusions of Jr(a+) RBCs to patients have resulted in no signs of hemolysis, although incompatible transfusion may cause a sharp rise in the titer of anti-Jra, resulting in signs of an acute HTR in subsequent transfusions. A patient with anti-Jra developed rigors after transfusion of 150 mL crossmatch-incompatible blood. Least-incompatible RBC units may be suitable for transfusion to most patients with anti-Jra, but Jr(a–) RBCs should be selected in cases where the anti-Jra is of high titer.2

LAN Blood Group System

LAN (Langereis) was officially recognized by the ISBT in 2012 as the 33rd human blood group system. It consists of one high-prevalence antigen, Lan (LAN1).25 The ABCB6 protein is the carrier of the Lan blood group antigen.16,25 The ABCB6 gene (chromosome 2q36, 19 exons) encodes the ABCB6 polypeptide, known as a porphyrin transporter. The exceptional Lan– individuals do not express ABCB6 (Lannull phenotype) owing to several different frameshift and missense mutations.25 To date, more than 40 ABCB6 alleles that encode Lan– or Lan+w phenotypes have been described,26 and quantitation of Lan antigen in Lan+, Lan+w, and Lan– phenotypes have been performed.27 Despite the Lan antigen role in erythropoiesis and detoxification of cells, Lan– individuals do not appear to demonstrate susceptibility to any disease.


Clinical SignificanceAnti-Lan has been reported in two African American

individuals as well as in other populations including Caucasians and Asians and may be stimulated by transfusion or pregnancy. The original anti-Lan was responsible for an immediate HTR characterized by fever and chills. There is no report of naturally occurring alloanti-Lan; none of the Lan– siblings of the Lan– propositi have anti-Lan. Anti-Lan has been described as having variable clinical significance, either for HTRs (none to severe) or HDFN (none to mild).25 Lan alloantibodies are mostly IgG1 and IgG3, although IgG2 and IgG4 may also be present. Some anti-Lan fix complement; others do not.2 Despite challenging conditions caused by the scarcity of Lan– donors worldwide, ideally Lan– RBCs should be selected for transfusion to patients with anti-Lan, especially individuals with a high-titer antibody,2,25 although least incompatible RBCs may be suitable for patients with weak examples of the antibody. The only autoanti-Lan was reported in a patient with mild autoimmune hemolytic anemia (AIHA) with depressed Lan antigen expression.2

Vel Blood Group System

Vel is an RBC antigen that is expressed by more than 99.9 percent of the population.28 The recognition of Vel dates to 1952, when a patient, named Mrs. Vel, suffered a transfusion reaction due to the presence of an antibody that was found to agglutinate sera of over 10,000 individuals. The antibody was named anti-Vel.29

Recently, the SMIM1 protein was shown to carry the Vel blood group antigen. Using a high-density single nucleotide polymorphism array, Storry et al.30 identified the SMIM1 gene residing in a 97-kb region of homozygosity on chromosome 1p36 in the vicinity of the RH locus. A frameshift deletion of 17 nucleotides in exon 3 of SMIM1 is responsible for the Vel– phenotype.16,31,32 Genotype screening estimated that ~1 in 17 Swedish blood donors is a heterozygous deletion carrier and ~1 in 1200 is a homozygous deletion knockout.31

Clinical SignificanceThe high clinical significance of Vel is related to what

happens to individuals with the Vel– phenotype upon transfusion or pregnancy. Vel alloantibodies are never naturally occurring, and most producers of anti-Vel have been transfused, yet Vel antibodies are predominantly IgM and fix complement. Of the two IgG anti-Vel, one was IgG1, and the other contained IgG1 and IgG3. Anti-Vel is a dangerous

antibody, and patients with anti-Vel should be transfused with Vel– RBCs. The first anti-Vel and other examples since have caused severe immediate HTRs. Anti-Vel may be missed in compatibility testing if inappropriate methods are used.33 Although many examples of anti-Vel have been found in pregnant women, anti-Vel does not usually cause HDFN, probably because most anti-Vel are predominantly IgM, and the Vel antigen is usually expressed weakly on neonatal RBCs.

Two examples of autoanti-Vel were responsible for (AIHA), although in one case, a nine-week-old infant, the RBCs gave a negative DAT.2

CD59

CD59, also known as the membrane inhibitor of reactive lysis (MIRL), homologous restriction factor (HRF), and membrane attack complex inhibitory factor (MACIF), is a cell surface glycoprotein of approximately 20 kDa that limits the activity of the terminal complement complex C5b-9 and is more effective than decay accelerating factor (DAF) or CD55 in this respect.28,34 The first demonstration of anti-CD59 was in a patient homozygous for a CD59 deficiency, which led to the discovery of a new blood group system, CD59, and a null allele (c.146delA).

CD59 is attached by a glycosylphosphatidylinositol (GPI) anchor not only to erythrocytes but also to various other cellular membranes. Seven cases of an isolated CD59 deficiency due to three distinct null alleles of the CD59 gene have been published so far.34 As well as being cell-bound through its GPI anchor, soluble CD59 is also found in plasma, urine, and cerebrospinal fluid.

Absence of CD59 is associated with hemolytic anemia and with thrombosis as well as with other autoimmune diseases such as systemic lupus erythematosus.2

Clinical SignificanceNo data are available.

Augustine Blood Group System

Ata is a high-prevalence antigen found on the RBCs of over 99 percent of individuals.35 The first literature referring to the Ata antibody, which can be responsible for severe HTRs and mild HDFN, dates to the 1960s. Applewhaite et al.36 identified an antibody with a novel specificity in the serum of Mrs. Augustine when the RBCs of her third child gave a positive DAT at birth. Her alloantibody, abbreviated as anti-Ata after



her name, reacted with more than 6600 blood donors tested at that time, indicating that Ata was a high-prevalence antigen. All anti-Ata producers reported thus far have been of African ancestry, like Mrs. Augustine.

Recently, it has been shown that SLC29A1 encoding the equilibrative nucleoside transporter 1 (ENT1) specifies a new candidate gene for a novel blood group system that includes the Ata antigen. Daniels et al.37 reported that a nonsynonymous SNP in SLC29A1 (rs45458701) is responsible for the At(a–) phenotype. Although all At(a–)-reported propositi are of African ancestry with functional ENT1, they identified three siblings of European ancestry who were homozygous for a null mutation in SLC29A1 (c.589+1G>C) and thus have the Augustinenull phenotype. These individuals lacking ENT1 exhibit periarticular and ectopic mineralization, which confirms an important role for ENT1/SLC29A1 in human bone homeostasis,34 cardioprotection, and drug transport in erythrocytes.38

Clinical SignificanceAta antibodies are mostly IgG, but IgM could also be

present and can directly agglutinate At(a+) RBCs. Of two IgG anti-Ata, one was IgG1, and the other consisted of IgG1, IgG3, and IgG4. Ata antibodies facilitate rapid destruction of 51Cr-labeled At(a+) RBCs in vivo and give positive results in the in vitro functional assay, the MMA. Ideally, At(a–) RBCs should be selected for transfusion to patients with anti-Ata, although least incompatible RBC units may be suitable for patients with weak examples of the antibody.2

One anti-Ata caused an immediate HTR with chills and nausea during an RBC survival study, and another caused a severe delayed HTR after transfusion of multiple units of At(a+) RBCs. Despite numerous pregnancies involving anti-Ata, only one of the infants had moderately severe HDFN requiring phototherapy.2 In three At(a–)-reported patients from the southern United States, the anti-Ata was concomitant with autoimmune disease.5

References

1. Crew VK, Burton N, Kagan A, et al. CD151, the first member of the tetraspanin (TM4) super family detected on erythrocytes is essential for the correct assembly of human basement membranes in kidney and skin. Blood 2003;102:4a.

2. Daniels G. Human blood groups. 3rd ed. London, UK: Wiley-Blackwell, 2013.

3. Hayes M. Raph blood group system. Immunohematology 2014;30:6–10.


4. Reid ME, Lomas-Francis C, Olsson ML. The blood group antigen factsbook. 3rd ed. San Diego, CA: Academic Press, 2012.

5. Johnson ST. JMH blood group system: a review. Immunohematology 2014;30:18–23.

6. Daniels G, Flegel WA, Fletcher A, et al. International Society of Blood Transfusion Committee on Terminology for Red Cell Surface Antigens: Cape Town report. Vox Sang 2007;92: 250–3.

7. Seltsam A, Agaylan A, Grueger D, et al. Rapid detection of JMH antibodies with recombinant Sema7A(CD108) protein and the particle gel immunoassay. Transfusion 2008;48:1151–6.

8. Bierhuizen MFA, Mattei MG, Fukuda M. Expression of the developmental I antigen by a cloned human cDNA encoding a member of a ß-1,6-N-acetylglucosaminyltransferase gene family. Genes Dev 1993;7:468–78.

9. Yu LC, Twu YC, Chang CY, et al. Molecular basis of the adult i phenotype and the gene responsible for the expression of the human blood group I antigen. Blood 2001;98:3840–5.

10. Inaba N, Hiruma T, Togayachi A, et al. A novel I-branching ß-1,6-N-acetylglucosaminyltransferase involved in the human blood group I antigen expression. Blood 2003;101:2870–6.

11. Hellberg A, Westman JS, Olsson ML. An update on the GLOB blood group system and collection. Immunohematology 2013;29:19–24.

12. Roudier N, Verbavatz JM, Maurel C, et al. Evidence for the presence of aquaporin-3 in human red blood cells. J Biol Chem 1998;273:8407–12.

13. Tilley L, Gren C, Poole J, et al. A new blood group system, RHAG: three antigens resulting from amino acid substitutions in the Rh-associated glycoprotein. Vox Sang 2010;8:151–9.

14. Barr K, Korchagina E, Popova I, et al. Monoclonal anti-A activity against the FORS1 (Forssman) antigen.Transfusion 2014;55:129–36.

15. Svensson L, Hult AK, Stamps R, et al. Forssman expression on human erythrocytes: biochemical and genetic evidence of a new histo-blood group system. Blood 2013;121:1459–68.

16. Storry J. Five new blood group systems: what next? ISBT Sci Ser 2014;9:136–40.

17. Castilho L, Reid ME. A review of JR blood group system. Immunohematology 2013;29:63–8.

18. Saison C, Helia V, Ballif BA, et al. Null alleles of ABCG2 encoding the breast cancer resistance protein define the new blood group system Junior. Nat Genet 2012;44:174–7.

19. Zelinski T, Coghlan G, Liu XQ, et al. ABCG2 null alleles define the Jr(a–) blood group phenotype. Nat Genet 2012;44:131–2.

20. Ogasawara K, Osabe T, Susuki Y, et al. A new ABCG2 null allele with a 27kb deletion including the promoter region causing the Jr(a–) phenotype. Transfusion 2015;55:1467–71.

21. Coghlan G. The JR blood group system: genetic and molecular investigations. ISBT Sci Ser 2012;7:260–3.

22. Hue-Roye K, Lomas-Francis C, Coghlan G, et al. The JR blood group system (ISBT 032): molecular characterization of the three new null alleles. Transfusion 2013;53:1575–9.

23. Fujita S, Kashiwagi H, Tomimatsu T, et al. Expression levels of ABCG2 on cord red blood cells and study of fetal anemia associated with anti-Jr(a). Transfusion 2016;56:1171–81.


24. Endo Y, Ito S, Ogiyama Y. Suspected anemia caused by maternal anti-Jr(a) antibodies: a case report. Biomark Res 2015;3:23.

25. Peyrard T. The LAN blood group system: a review. Immunohematology 2013;29:131–5.

26. Reid ME, Hue-Roye K, Huamg A, et al. Alleles of the LAN blood group system: molecular and serologic investigation. Transfusion 2014;54:398–404.

27. McBean R, Wilson B, Liew Y, et al. Quantitation of Lan antigen in Lan+, Lan+w, and Lan– phenotypes. Blood Transfus 2015; 13:662–5.

28. Hayer-Wigman I, deHaas M, van der Schoot CE. The immune response to the VEL antigen is HLA class II DRB1*11 restricted. Vox Sang 2013;105(suppl 1):29.

29. Race RR, Sanger R. Blood groups in man. 6th ed. Philadelphia, PA: Blackwell Science Ltd., 1975:413.

30. Storry JR, Joud M, Christophersen MK, et al. Homozygosity for a null allele of SMIM1 defines the Vel-negative blood group phenotype. Nat Gen 2013;45:537–41.

31. Vejic A, Haer-Wigman L, Stephens JC, et al. SMIM1 underlines the Vel blood group and influences red blood cell traits. Nat Genet 2013;45:542–5.

32. Ballif BA, Helia V, Peyrard T, et al. Disruption of SMIM1 causes the Vel– blood type. EMBO Mol Med 2013;5:751–61.

33. Storry JR, Mallory D. Misidentification of anti-Vel due to inappropriate use of techniques. Immunohematology 1994;10: 83–6.

34. Anliker M, Zabera I, Hochsmann B, et al. A new blood group antigen is defined by anti-CD59 detected in a CD59-deficient patient. Transfusion 2014;54:1817–22.

35. McBean R, Liew Y, Wilson B, et al. Genotyping confirms inheritance of the rare At(a–) type in a case of hemolytic disease of the newborn. J Path Clin Res 2016;2:53–5.

36. Applewhaite F, Ginsberg V, Gerena J, et al. A very frequent red cell antigen Ata. Vox Sang 1967;13:444–5.

37. Daniels G, Ballif B, Helias V, et al. Lack of the nucleoside transporter ENT1 results in the Augustine-null blood type. Blood 2015;125:3651–4.

38. Rose JB, Naydenova Z, Bang A, et al. Equilibrative nucleoside transporter 1 plays an essential role in cardioprotection. Am J Physiol Heart Circ Physiol 2010;298:H771–7.

Mostafa Moghaddam, MA, CLS(ASCP)BB, Head of Immunohematology Reference Laboratory, (corresponding author), Department of Immunohematology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Iranian Blood Transfusion Organization, IBTO Tower, Hemat Expressway, Tehran, Iran, [email protected]; and Amir Ali Naghi, DMT, Senior Technologist, Department of Immunohematology, Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Iranian Blood Transfusion Organization, Tehran, Iran.

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Rouleaux and saline replacementK.L. Waider

Rouleaux is a phenomenon that commonly occurs in patients who have an increased number of circulating protein macromolecules. It is a benign, in vitro reaction that appears microscopically as red blood cells (RBCs) line up against each other; many liken the RBC aggregation to “stacked coins.” This unexpected reactivity may cause confusion in direct agglutination testing such as reverse blood typing and crossmatching. Saline replacement is the established method to resolve rouleaux. True agglutination will remain when plasma is replaced with saline for resuspension of the RBC button. Rouleaux will no longer be seen when the plasma proteins are removed. Immunohematology 2018;34:91–92.

Key Words: rouleaux, stacked coins, saline replacement, macromolecule

Principle

Rouleaux is a type of red blood cell (RBC) aggregation with a distinct “stacked coins” appearance when observed microscopically. These aggregates frequently have a more refractive appearance than classic RBC agglutination. There are typically many “stacks” in the sample, and their size can vary greatly. The rouleaux aggregates disperse differently from the main RBC button during resuspension, such that experienced technologists can sometimes recognize this phenomenon even before confirming it microscopically. It is most typically seen in patients with disease states that cause an increase in plasma protein macromolecules, such as multiple myeloma, diabetes mellitus, and many infections.1 The macromolecules are thought to cause rouleaux by two unrelated mechanisms: the bridging model or the depletion model. One or both may occur in an individual. In the bridging model, the macromolecules partially adsorb into the RBC and bridge to the RBC closest to it. In the depletion model, the macromolecules move away from the RBCs and change the osmotic pressure between RBCs causing them to aggregate (Fig. 1).2 Once recognized, rouleaux can be resolved using the saline replacement method. By replacing the patient’s plasma/serum with saline, the macromolecules are removed, and the RBCs dissociate.

Indications

Rouleaux may be present in any direct agglutination test containing plasma or serum. Examples are reverse ABO typing, immediate spin crossmatch, and immediate spin and/or 37°C reading of antibody detection and/or identification testing. If unexpected positive results present after routine incubation and centrifugation, rouleaux should be considered. Once confirmed, the saline replacement method3 should be performed using the tube method. It is important to remember that rouleaux can only be found in the presence of patient plasma or serum, since this is where the interfering macromolecules are found. Rouleaux is not seen in the antiglobulin phase of testing because the wash step rids the test system of the patient’s plasma/serum.

Procedure

Examine suspected reactivity microscopically. Rouleaux will appear as stacked coins, whereas true agglutinates are

Reagents/Supplies

Reagents Supplies

• 0.9% saline or PBS (pH 6.5–7.5)

• Transfer pipettes

• 10 × 75 or 12 × 75 mm tubes

• Calibrated serologic centrifuge

• Microscope

PBS = phosphate-buffered saline.

Procedural Steps

• Confirm the presence of rouleaux microscopically following initial RBC resuspension.

• Re-centrifuge the test tube.• Remove patient’s plasma/serum using transfer pipettes.• Replace with saline.• Resuspend. Confirm.

RBC = red blood cell.

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K.L. Waider

amorphous clumps. After confirming the presence of rouleaux, re-centrifuge the plasma (or serum) and cell mixture. Carefully remove the plasma using transfer pipettes, leaving the RBC button undisturbed. This effect can be achieved by tilting the tube so that the plasma is away from the cell button, allowing the pipette to aspirate it without touching the RBCs. Gently replace the plasma with an equal volume of saline without disturbing the RBC button. Resuspend the RBC button, and observe for agglutination. Rouleaux will disperse when suspended in saline, but true agglutination is stable in the presence of saline and will remain.

Once saline replacement has been performed, only direct agglutination testing may be interpreted. Serum/plasma may not be reintroduced to the test system. If test reading at additional phases are desired, the test must be repeated using a separate tube for each phase where a reading will occur.

Limitations

Rouleaux does not typically cause interference in column testing, although high protein levels can lead to false positives or hazy reactions in this method.4 Confirmation of rouleaux and resolution must be performed with the tube method.

Acknowledgments

The author would like to thank Jan Hamilton for her guidance while working on this manuscript.

References

1. Samsel RW, Perelson AS. Kinetics of rouleau formation. I. A mass action approach with geometric features. Biophys J 1982;37:493–514.

2. Bäumler H, Neu B, Donath E, et al. Basic phenomena of red blood cell rouleaux formation. Biorheology 1999;36:439–42.

3. Fung MK, Ed. Method 3-7: Detecting antibodies in the presence of rouleaux-saline replacement. In: Technical manual. 19th ed. Bethesda, MD: AABB, 2017.

4. Blood grouping reagent MTS A/B/D monoclonal and reverse grouping card [package insert]. Pompano Beach, FL: Micro Typing Systems Inc., 2008.

Kayla L. Waider, BS, MLS(ASCP)CM, Immunohematology Reference Laboratory Technologist III, American Red Cross Biomedical Services, Southeastern Michigan Region, 100 Mack Avenue, Detroit, MI 48201, [email protected].

Fig. 1 (A) Schematic drawing of the bridging model. The adsorption of dextran into single cells and the formation of bridges during the absence of shear flow lead to rouleaux formation of cells. (B) Schematic drawing of the depletion model. Depletion of macromolecules from the interface (with or without a weak adsorption) leads to rouleaux formation if the cells come into close proximity due to the osmotic pressure difference between the pressure in the bulk phase and in the gap. (Reprinted from Bäumler et al.2 with permission from Biorheology.)

(A)

(B)


Method-specific and unexplained reactivity in automated solid-phase testing and their association with specific antibodiesM.E. Harach, J.M. Gould, R.P. Brown, T. Sanders, and J.H. Herman

ORiginal RepORt

The inherent tradeoff between sensitivity and specificity in the detection of unexplained antibodies has been the objective of many studies, editorials, and journal articles. Many publications note that no method is capable of detecting all clinically significant antibodies while avoiding all clinically insignificant antibodies. This study describes the frequency of nonspecific reactivity and unexplained reactivity in solid-phase testing, along with the subsequent development of specific antibodies (Abs). In this study, nonspecific reactivity (NS) is defined as method-specific panreactivity detected by solid-phase testing only, with no reactivity in other methods. Unexplained reactivity (UR) is defined as reactivity present and detectable in all test methods after all clinically significant antibodies were ruled out following a standard antibody identification algorithm using selected cell panels. This retrospective study evaluated antibody detection tests of patients at a single center for 2 years using two automated solid-phase instruments that used the same three-cell antibody detection test. Antibody identification was performed with solid-phase panels supplemented with a polyethylene glycol tube method as needed. Of the 1934 (5%) samples with a positive antibody detection test, 29 had unavailable work-up data, leaving 1905 (98.5%) samples eligible for inclusion in the study. The data revealed the following: Ab only 999 (52.4%); UR only 429 (22.5%); Ab and UR 227 (11.9%); NS only 206 (10.8%); Ab and NS 24 (1.3%); UR and NS 14 (0.7%); and Ab, UR, and NS 6 (0.3%). Patients with a positive follow-up antibody detection test had UR and NS replaced with a specific Ab in 23 of 656 UR (3%) and 8 of 230 NS (3%) cases, respectively. Additionally, six patients with UR developed a specific Ab along with persistent UR, and no patients with persistent NS developed a specific Ab. The study concluded that both UR and NS can be encountered in solid-phase testing, and both UR and NS can persist in follow-up testing. Specific Ab was observed to replace UR in a few patients. Immunohematology 2018;34:93–97.

Key Words: immunohematology, red blood cell serology, blood groups, nonspecific reactivity, unexpected reactivity

Transfusion medicine professionals have the responsibility to identify and provide compatible blood components for transfusion to their patients. This responsibility includes the detection and identification of unexplained antibodies. According to the 18th edition of the AABB Technical Manual, the goals of antibody detection are to detect in a timely manner

as many clinically significant antibodies as possible while avoiding as many clinically insignificant antibodies.1

This inherent tradeoff between sensitivity and specificity in the detection of unexplained antibodies has been the objective of many studies, editorials, and journal articles. Although all these publications note that no method is capable of detecting all clinically significant antibodies while avoiding all clinically insignificant antibodies, some of these studies reported decreased detection of insignificant antibodies by gel,2 and others noted the superior detection of clinically significant antibodies by solid-phase testing.3 In their study, Liu and Grossman4 noted that in gel an “antibody of undetermined specificity (AUS) was reported 1442 times (18%) and was the single most reported event” of the 8121 antibodies evaluated. In the same study, they document that in patients presenting with AUS for the first time, the majority of AUS persist in subsequent workups and that some AUSs even develop into clinically significant antibodies.4 The 2017 study by Miller et al.5 noted the increased sensitivity of general nonspecific reactivity in solid-phase testing when compared with tube method testing using polyethylene glycol (PEG) and recommends this nonspecific reactivity to not be ignored because of the noteworthy proportion of these antibodies that develop into specific and clinically significant antibodies. The Miller et al. study, however, did not focus on the frequency with which nonspecific reactivity might progress to the level of true antigen specificity, as did the Liu and Grossman study of the gel method. In our current study, nonspecific reactivity (NS) is defined as method-specific panreactivity detected by solid-phase testing only, with no reactivity in other methods. This reactivity is in contrast to unexplained reactivity (UR), defined as reactivity present and detectable in all test methods after all clinically significant antibodies were ruled out following our standard antibody identification algorithm using selected cell panels.6 Our data analysis attempts to enumerate the frequency of UR and NS as well as evaluate their association with development of specific antibodies (Abs) over time.


Materials and Methods

This retrospective study evaluated the antibody detection test results of patients between 1 January 2012 and 31 December 2013. In this single facility, for the time period under evaluation, all antibody detection tests were performed on two different automated instruments (Echo and Galileo; Immucor, Norcross, GA). Both instruments use the same three-cell antibody detection test plates (Capture-R Ready-Screen, Immucor) to reduce ambiguity between donor cells. Antibody detection test results were read and graded by the automated instruments or by a medical laboratory scientist who read and graded (negative, weak+, 1+, 2+, 3+, or 4+) results according to method 1–9 in the AABB Technical Manual.1

In the instance of a positive antibody detection test result, the patient’s historical antibody records were reviewed in addition to the ensuing antibody identification performed on the current sample. Antibody identification was performed primarily with solid-phase panels (Capture-R Ready-ID, Capture-R Ready-ID Extend I and Extend II; Immcor). If needed, this testing was supplemented with a PEG additive (Gamma PeG, Immucor) tube method using a 10-minute incubation. Antibody reactions were read and graded by the same methods as previously described.

An observation of UR or NS resulted in reviewing the past records of the associated patient and evaluating subsequent samples for the presence of UR and/or NS and for the development of specific Abs. Based on the results of the follow-up testing of these subsequent samples, patients were assigned to one of the following categories:

• Category 1: No testing performed• Category 2: Positive antibody detection test; antibody

identification unresolved• Category 3: Negative antibody detection test• Category 4: Positive antibody detection test; UR or NS

replaced by specific Ab• Category 5: Positive antibody detection test; UR or NS

only remained

Results

In the 24-month study period, antibody detection testing was performed on a total of 38,178 patients. Of these, 1934 (5%) had a positive antibody detection test. The antibody identification workup was unavailable for 29 of these patients, leaving 1905 (98.5%) eligible for inclusion in this study.

Results of antibody identification testing revealed UR was present in 917 of 1905 (48%) patients, and NS was present in 250 of 1905 (13%) patients. The various combinations of Ab, UR, and NS were as follows: Ab only 999 (52.4%); UR only 429 (22.5%); Ab and UR 227 (11.9%); NS only 206 (10.8%); Ab and NS 24 (1.3%); UR and NS 14 (0.7%); and Ab, UR, and NS 6 (0.3%). For the samples in which UR and NS were both noted, UR was detected in a different antibody identification method (PEG tube) after NS was detected in the solid-phase method. The results are summarized in Table 1. There was a history of a specific Ab in 141 of 656 (21%) patients with UR and in 42 of 230 (18%) patients with NS.

For samples in which UR or NS was observed, follow-up testing of subsequent samples was evaluated, and the patients were assigned to one of the following categories:

• Category 1: No testing performed in 326 of 656 UR (50%) and 129 of 230 NS (56%)

• Category 2: Positive antibody detection test; antibody identification unresolved in 89 of 656 UR (14%) and 51 of 230 NS (22%)

• Category 3: Negative antibody detection test in 178 of 656 UR (27%) and 23 of 230 NS (10%)

• Category 4: Positive antibody detection test; UR or NS replaced by specific Ab in 23 of 656 UR (3%) and 8 of 230 NS (3%)

• Category 5: Positive antibody detection test; UR only remained in 40 of 656 UR (new specific antibody developed in 6 of 40 of these) or NS only in 19 of 230 NS

Category 4 and 5 results are graphically depicted in Figure 1.

M.E. Harach et al.

Table 1. Solid-phase antibody evaluation summary

Antibody work-up results (N = 1905)

n %*

Antibody only 999 52.4

Unexplained reactivity only 429 22.5

Antibody and unexplained reactivity 227 11.9

Nonspecific reactivity only 206 10.8

Antibody and nonspecific reactivity 24 1.3

Unexplained reactivity and nonspecific reactivity 14 0.7

Antibody, unexplained reactivity, and nonspecific reactivity 6 0.3

*Percent of total, rounded to nearest 0.1%.


Method-specific and unexplained reactivity

Discussion

Our data also shed light on the constant balance between sensitivity and specificity. During follow-up testing, we determined the development from UR and/or NS to true Ab specificity. Those samples in categories 1 and 2 can be omitted because of either a lack of follow-up or no resolution of specific antibody identification. Of those samples with evaluable follow-up antibody detection and identification in categories 3, 4, and 5, UR was undetectable in 201 of 241 (83%) and persisted in

40 of 241 (17%), and NS was undetectable in 31 of 50 (62%) and persisted in 19 of 50 (38%). Specific Ab development in patients in categories 3, 4, and 5 occurred in 29 of 241 (12%) of previous UR and 8 of 50 (16%) of NS. Figure 1 depicts the specific Abs identified in place of the UR/NS.

Of interest is that during the study period, the manufacturer (Immucor) alerted users to an increase in UR reported to them by their customers. Steps were taken during reagent manufacturing, and resolution of the increase in UR was communicated to users based on customer reports. To

Fig. 1 (A) Unexplained reactivity (UR) replaced with antibodies and reactivity, as shown. (B) Nonspecific reactivity (NS) replaced with antibodies and reactivity, as shown. HLA = human leukocyte antigen.

Solid-phase nonspecific reactivity (NS)

HLA antibody

Cold antibody

Anti-Lea

Anti-K

Anti-Jkb

Anti-D

Anti-c

Anti-E

Anti-Jka

10 2 3 4 5 6 7 8

Number of patients

A

UR

Warm autoantibody and UR

Warm autoantibody

Cold antibody

Anti-Jkb and UR

0 1 2 3 4

Number of patients

B


determine whether the UR alert affected UR and NS rates at our facility, the study periods before, during, and after the alert period were compared. Based on the issuance date of the alert (22 March 2013), the pre-alert period was defined as 1 January 2012 through 30 September 2012, the alert period was defined as 1 October 2012 through 30 April 30 2013, and the post-alert period was defined as 1 May 2013 through 31 December 2013.

A decrease in the rate of UR and NS after the alert period was noted on the smaller instrument (Echo, Immucor) (Fig. 2). There was a 26 percent overlap of patients with antibody detection performed on either instrument (Galileo or Echo, Immucor) on different dates, but because of the retrospective nature of the study and the relatively small numbers, no further trend was apparent in distinguishing UR and NS on the two solid-phase devices. Further studies are in progress at this facility to fully evaluate the impact of the improvements communicated on the UR and NS rates. Additionally, these subsequent studies use a newer instrument (Galileo NEO, Immucor) and will generate data as a second phase to this original study with the updated automated instrument.

Conclusions

The patients who have evaluable follow-up antibody detection testing are described in categories 3, 4, and 5. Based on analysis of the testing performed on samples from these

patients, it can be concluded that UR and NS antibodies are encountered in solid-phase testing and may be transient. In these patients, the UR was undetectable in 201 of 241 (83%) and persisted in 40 of 241 (17%) of the patients on follow-up testing. Also, the NS was undetectable in 31 of 50 (62%) and persisted in 19 of 50 (38%) of the patients upon follow-up testing.

UR and NS antibodies detected in solid-phase testing may develop into true antibody specificity. Of those patients with evaluable follow-up antibody detection tests (categories 3, 4, and 5), a new antibody developed in 29 of 241 patients with a previous UR (12%) and in 8 of 50 patients with a previous NS (16%). Based on a high regard for patient safety, which includes the maximum detection of clinically significant antibodies, this facility has selected the automated solid-phase method—knowing the rate of UR and NS—because of the well-documented superior sensitivity of Capture-R3,7 compared with other testing methods such as tube testing.

References

1. Fung MK, Grossman BJ, Hillyer CD, Westhoff CM. Technical manual. 18th ed. Bethesda, MD: AABB, 2014.

2. Judd WJ, Steiner EA, Knafl PC. The gel test: sensitivity and specificity for unexplained antibodies to blood group antigens. Immunohematology 1997;13:132–5.

3. Weisbach V, Kohnauser T, Zimmermann R, et al. Comparison of performance of microtube column systems and solid-phase systems and the tube low-ionic-strength solution

M.E. Harach et al.

Fig. 2 The frequency of unexplained reactivity (UR)/nonspecific reactivity (NS) changed during the study period, which overlapped with the manufacturer’s alert regarding UR/NS. The pre-alert period is defined as 1 January 2012 to 30 September 2012, the alert period is 1 October 2012 to 30 April 2013, and the post-alert period is 1 May 2013 to 31 December 2013.

Echo UR

Echo NS

Galileo UR

Galileo NS

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0

Perc

ent

Pre-alert Alert Post-alert


Method-specific and unexplained reactivity

additive indirect antiglobulin test in the detection of red cell alloantibodies. Transfus Med 2006;16:276–84.

4. Liu C, Grossman BJ. Antibody of undetermined specificity: frequency, laboratory features, and natural history. Transfusion 2013;53:931–7.

5. Miller NM, Johnson ST, Carpenter E, Naczek CA, Karafin MS. Patient factors associated with unidentified reactivity in solid-phase and polyethylene glycol antibody detection methods. Transfusion 2017;57:1288–93.

6. US Food and Drug Administration. Subpart D, Reagent Red Blood Cells. Testing of source material. 21 CFR Sect. 660.33 (2017). eCFR Code of Federal Regulations. Available from https://www.ecfr.gov/cgi-bin/text-idx?SID=26cb4151fc77996051b7706a77bcb400&mc=true&node=pt21.7.660&rgn=div5#se21.7.660_133.

7. Kay B, Poisson JL, Tuma CW, Shulman IA. Anti-Jka that are detected by solid-phase red blood cell adherence but missed by gel testing can cause hemolytic transfusion reactions. Transfusion 2016;56:2973–9.

Mary E. Harach, BS, MT(ASCP), Quality Assurance Coordinator (corresponding author), Thomas Jefferson University Hospital, Department of Transfusion Medicine, 111 South 11th Street, Suite 8250 Gibbon, Philadelphia, PA 19107, [email protected]; Joy M. Gould, BB(ASCP)C, SBB, Blood Bank Supervisor, Thomas Jefferson University Hospital, Department of Transfusion Medicine, Philadelphia, PA; Rosemary P. Brown, MT(ASCP), Senior Medical Technologist–Blood Bank, Thomas Jefferson University Hospital, Department of Transfusion Medicine, Philadelphia, PA; Tricia Sanders, MBA, MLS(ASCP)CMSBBCM, Global Education and Technical Marketing Program Manager, Immucor Diagnostics, Norcross, GA; and Jay H. Herman, MD, Thomas Jefferson University Hospital, Department of Pathology, Anatomy and Cell Biology, Philadelphia, PA.

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Utility of chloroquine diphosphate in the blood bank laboratoryT. Aye and P.A. Arndt

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Chloroquine diphosphate (CDP) is a helpful tool in the blood bank for two main applications. The most common application is to render direct antiglobulin test–positive red blood cells (RBCs) free from membrane-bound IgG; these treated RBCs can then be used for autologous adsorption and/or to determine the patient’s RBC phenotype. Another common use of CDP is to remove human leukocyte antigens (HLAs) from RBCs to help identify or exclude the presence of antibodies to HLAs expressed on RBCs, for example, Bennett-Goodspeed (Bg) antigens. In this review, the principles, applications, and limitations of using CDP are discussed. Immunohematology 2018;34:98–102.

Key Words: chloroquine diphosphate, direct antiglobulin test, human leukocyte antigen

Antigen typing of red blood cells (RBCs) using an indirect antiglobulin test (IAT) method can be challenging when RBCs are coated with IgG antibodies and thus have a positive direct antiglobulin test (DAT). Commercially available monoclonal antisera that can be used at the direct agglutination phase without an IAT are not available for typing of all blood group antigens. For those monoclonal antisera that are available, false-positive and false-negative results can occur when testing untreated RBCs if they are strongly coated with IgG.1–3 The majority of “in-house” or “rare” antiserum is collected from patients and is usually reactive only by the IAT. When there are no commercially available antisera that can be used by direct agglutination tests and/or the patient’s sample has a strongly positive DAT, there is a need for a reliable method to dissociate membrane-bound IgG to then obtain an accurate RBC phenotype using an IAT method.

Although chloroquine diphosphate (CDP) was reported in the 1970s to remove warm autoantibodies from DAT+ RBCs,4,5 its use was not applied to antigen-typing of RBCs with a positive DAT until the 1980s (by Edwards et al.6) There were only two other procedures available at that time—gentle heat elution (e.g., 45°C for 10–30 minutes or 56°C for 3 minutes)7 or treatment with ZZAP, a combination of dithiothreitol (DTT) and papain.8 Heating RBCs above 37°C weakens antigens relative to the temperature and time of incubation9 and ZZAP destroys antigens sensitive to DTT and papain (Kell, Duffy,

Reagents/Supplies — Room Temperature

Reagents Supplies

• Anti-IgG

• IgG-coated RBCs

• 16–20% CDP solution at pH 5.0 ± 1

• Isotonic saline

• Pipettes

• Test tubes: 10 × 75 or 12 × 75 mm

• Calibrated centrifuge

RBCs = red blood cells; CDP = chloroquine diphosphate.

Procedural Steps — Room Temperature

• Mix 1 volume of washed, packed RBCs with 4 volumes of 16–20% CDP.

• Incubate mixture at room temperature.• At 30-minute intervals, wash a small aliquot of RBCs at least three

times, and perform DAT using anti-IgG to monitor the process of IgG dissociation. The mixture can be incubated for up to 2 hours.

• When the DAT is negative, wash the remaining CDP-treated RBCs at least three times with isotonic saline.

RBCs = red blood cells; CDP = chloroquine diphosphate; DAT = direct antiglobulin test.

Reagents/Supplies — 37°C

Reagents Supplies

• Anti-IgG

• IgG-coated RBCs

• 16–20% CDP solution at pH 5.0 ± 1

• Isotonic saline

• Pipettes

• Test tubes: 10 × 75 or 12 × 75 mm

• Calibrated centrifuge

• 37°C water bath or heat block

RBCs = red blood cells; CDP = chloroquine diphosphate.

Procedural Steps — 37°C

• Mix 1 volume of washed, packed RBCs with 4 volumes of 16–20% CDP.

• Incubate mixture at 37°C.• At 5-minute intervals, wash a small aliquot of RBCs at least three

times and perform a DAT using anti-IgG to monitor the process of IgG dissociation. The mixture can be incubated for up 30 minutes.

• When DAT is negative, wash the remaining CDP-treated RBCs at least three times with isotonic saline.

RBCs = red blood cells; CDP = chloroquine diphosphate; DAT = direct antiglobulin test.


Use of CDP

MNS, etc.). Gentle heat elution and ZZAP are good methods for preparing RBCs for autoadsorption; CDP has also been described for this use.

Treatment of DAT+ RBCs with glycine-HCl/EDTA (also known as EDTA glycine acid [EDTA acid]) was described by Louie et al.10 and reviewed by Kosanke.11 It is quick and reliable but destroys Kell system antigens and Era. Microwave irradiation was also described to remove IgG from RBCs and allow phenotyping.12 All these methods have their limitations. Most significant is the potential for weakening or destroying RBC antigens when the treatment is done to prepare RBCs for phenotyping. Because of these limitations, there is a need to use more than one method in an immunohematology reference laboratory. Depending on the antigens of interest, an appropriate method can be selected to dissociate membrane-bound IgG from DAT+ RBCs.

Bennett-Goodspeed (Bg) antigens are human leukocyte antigen (HLA) class I (HLA-A and HLA-B) weakly expressed on RBCs, including the reagent RBCs used in blood bank testing. Despite their clinical insignificance, Bg antibodies may interfere in routine antibody detection and identification tests by showing weak unexpected reactions without a distinct pattern. CDP and EDTA acid destroy Bg antigens on RBCs; thus, these reagents can be useful in identifying or excluding reactions caused by Bg antibodies.13,14

Principle

CDP diluted to 16–20 percent (wt/vol) at a final pH of 5.0 ± 0.1 is used to dissociate membrane-bound IgG from RBCs.6,15 Issitt and Anstee16 suggested that the most likely mechanism for this dissociation involves neutralization of charged groups on amino acids that influence the shape of the antibody molecules that bind to antigens. Edwards et al.6 found that CDP was effective on 84 percent of in vivo sensitized DAT+ samples tested; results of other studies are summarized in Table 1. Factors that influence the ability of CDP to dissociate antibodies from DAT+ RBCs may be the IgG subclass of the antibody, the antibody affinity, the accessibility of the antigen-antibody complex, and the quantity of antibody bound to the RBCs.6 The temperature of CDP treatment (e.g., room temperature versus 37°C) and the sensitivity of the DAT method (e.g., tube versus gel) can also affect the outcome of CDP treatment.

CDP can remove HLAs expressed on RBCs (e.g., Bg antigens).13,14 This fact can be useful in antibody detection and identification tests where the antibodies may show weak reactivity with no apparent specificity. By testing a patient’s plasma with CDP-treated RBCs in parallel with untreated RBCs, the presence of Bg antibodies may be identified presumptively. The Bg antibodies may then be confirmed

Table 1. Studies on the efficiency of CDP for clearing IgG versus other methods

Reference

IgG-coated RBC samples

Results using RT or 37°C CDP method Results by other methodsNumber Type* Strength

6 56 In vivo 1–4+ RT = 47 (84%) neg NA

40 In vitro 3–4+ RT = 22 (55%) neg NA

10 30 In vivo Strong+ RT = 26 (87%) neg EDTA acid = 30 (100%) neg; ZZAP = 28 (93%) neg

12 15 In vivo 1–3+ RT = 4 (27%) neg Microwave = 7 (47%) neg

17 20 In vitro Variable RT = 10 (50%) neg37°C = 20 (100%) neg

ZZAP = 18 (90%) neg

18 8

42

In vivo

In vitro

1–4+ RT = 26 (52%) neg by tube, 22 (44%) neg by gel

EDTA acid = 48 (96%) neg by tube, 42 (84%) neg by gel

56°C heat = 46 (92%) neg by tube, 25 (50%) neg by gel

19 26 In vivo ±–4+ RT = 25 (96%) neg EDTA acid = 25 (96%) neg

20 93 In vivo w+–4+ RT = 29 (31%) â by gel EDTA acid = 59 (63%) â by gel56°C heat = 36 (39%) â by gel

17 In vitro No data available RT = 5 (29%) neg by gel EDTA acid = 12 (71%) neg by gel56°C heat = 9 (53%) neg by gel

*In vivo = direct antiglobulin test (DAT)+ red blood cells (RBCs); In vitro = RBCs coated with antibody in the laboratory.CDP = chloroquine diphosphate; RT = room temperature; neg = negative; NA = not applicable; â = reduction in strength.


or excluded by further testing with routine HLA laboratory methods or by testing with RBCs known to show strong expression of Bg antigens. Along with Bg, CDP may destroy or weaken other important RBC antigens (see Limitations). Because of the possible effect that CDP can have on these other antigens, caution must be taken in antibody exclusions when identifying interfering reactions due to Bg antibody using CDP-treated RBCs. Other blood group antigens reported to be affected by CDP are summarized in Table 2.

Procedural Steps

The supplies and procedural steps for CDP treatment are provided. If commercially manufactured CDP reagent is

used, the manufacturer’s recommendations supersede what is described here.

The most common method involves CDP treatment at room temperature with an incubation time that varies from 30 to 120 minutes.6,15 Briefly, 4 volumes of 16–20 percent chloroquine, pH 5.0 ± 0.1, are added to 1 volume of washed, packed DAT+ RBCs. After 30 minutes, a small subsample is removed and washed, and a DAT is performed using anti-IgG. If the DAT is negative, then the rest of the sample can be washed and used for phenotyping and/or autoadsorption. If the DAT is still positive, then the incubation can continue for another 30 minutes and the process is repeated (to a maximum incubation time of 2 hours). An alternative method involves CDP treatment at 37°C.17,24,29 Beaumont et al.24 studied RBC

T. Aye and P.A. Arndt

Table 2. Blood group antigens reported to be significantly affected or not affected by CDP

Blood group system/ antigen Significantly affected by CDP Not affected by CDP

ABH NDA (NI)6,13

Rh (D, C, e; reduced and saline antisera)21; (D; direct testing, few by IAT)22; (D, C, E, c, e, Cw; weakened at RT and 37°C)17

(NI)6,13,20; (C)23; (D, C, E, c, e at 37°C)24; (C, c, E, e)18

MNS (Mta)25 (NI)6,13,20; (S, s)18,19,26; (S)27; (Mg, Mta, He)13; (M, N, S, s at RT and 37°C)17

P1PK (P1 weakened at 37°C)17 (NI)6; (P1)13; (P1 at RT)17

Lewis (Lea, Leb, weakened at 37°C)17 (NI)6,13; (Lea, Leb at RT)17

Kell NDA (NI)6,13,20; (K, k)26; (K, k, Kpa, Kpb at RT and 37°C)17; (K, k at 37C)24; (K, k, Kpb)18; (K, Kpa)2

Duffy (Fyb)26; (Fyb, sig â after 2 hours)27; (NI, weakened)20 (NI)6,13; (Fya, Fyb)18,19,26; (Fya)2,27; (Fya, Fyb at RT and 37°C)17; (Fya, Fyb at 37°C)24

Kidd (Jkb weakened after 2 hours at RT, Jka weakened at 37°C)28; (Jkb at 37°C)24

(NI)6,13,20; (Jka, Jkb)18,19,26–28; (Jka, Jkb at RT and 37°C)17; (Jka at 37°C)24

Knops (NI)25 (Kna)27; (Kna, McCa, Yka)13; (Kna, McCa, Sla, Yka)23

Lutheran (Lub)26; (Lub, slight â)27 (Lub)26

JMH (JMH)26; (JMH, slight â)27 (JMH)13

Vel (Vel, sig â after 120 minutes)27 NDA

Yt (Yta)26; (Yta, sig â after 2 hours)27 (Yta)13,26

Chido/Rodgers NDA (Ch, Rg)13

Lan NDA (Lan)13

Cromer NDA (Cra)13

Colton NDA (Coa)13

Dombrock (NI)25 (Gya)13

Diego NDA (Bpa, Rba, Wda, Wra, Vga)13

Indian NDA (Ina)13

Cost NDA (Csa)13,23

700 Series NDA (Bya)13

Miscellaneous NDA (Cad)13

CDP = chloroquine diphosphate; NDA = no data available; NI = specificities not indicated; IAT = indirect antiglobulin test; RT = room temperature; sig = significant; â = decrease.


treatment with CDP at 18°C, 25°C, 30°C, and 37°C. Incubation at 37°C is known to facilitate IgG elution; when incubating at 37°C, a subsample is removed and washed, and a DAT is performed using anti-IgG every 5 minutes (to a maximum incubation time of 30 minutes).24 In both methods of treatment (room temperature or 37°C), there is a threshold for incubation time where further incubation will not facilitate dissociation of antibody from RBCs, and the RBCs start to deteriorate. An alternative treatment method to dissociate IgG should be considered.

For removal of Bg antigens, Swanson and Sastamoinen13 and Champagne et al.14 treated RBCs with commercially available chloroquine (Gamma-Quin; now available from Immucor, Norcross, GA) at room temperature for 90 minutes13 or up to 2 hours.14

Indications

CDP treatment can be used when a patient’s RBCs are coated with IgG and there is a need to perform antigen typing using antisera that are reactive only at the IAT phase and/or to prove the presence of an autoantibody by testing a patient’s plasma, serum, or eluate against DAT– autologous RBCs. Treatment with CDP dissociates membrane-bound IgG, allowing the treated RBCs to be tested at the IAT phase without interference. CDP treatment can also be used to avoid false-positive or false-negative results with monoclonal antisera when testing a patient’s RBCs that are strongly coated with IgG. For institutions that advocate the use of specific antigen–matched blood for transfusion in the presence of a warm autoantibody, an RBC phenotype from DAT+ RBCs can be obtained by treating the RBCs with a chemical such as CDP, as long as the patient has not been recently transfused. CDP treatment can also be used to remove or reduce RBC-bound IgG so the patient’s RBCs can be used in autologous adsorptions (again, as long as the patient has not been recently transfused).

When Bg antibodies are suspected to cause weak interfering reactions in antibody detection or identification testing and a laboratory does not have the capability to identify HLA class I antibodies in a patient’s plasma, CDP-treated cells can be tested in parallel with untreated RBCs to presumptively exclude or identify Bg antibodies and to allow the identification of other RBC alloantibodies. The abrogation of reactivity with CDP-treated RBCs suggests that the weak interfering reactions may be caused by Bg antibodies.

Limitations

Weakening or denaturation of some RBC antigens was observed in some instances (Table 2). Per the manufacturer’s instructions for Gamma-Quin,15 CDP-treated RBCs can be typed with high-protein reagents or those reactive by IAT but should not be typed with saline-reactive or monoclonal blood typing reagents because of weaker-than-expected reactions. Mallory and Reid26 reported false-negative antigen typing of some CDP-treated RBCs when tested with some antiglobulin reacting antisera. McShane and Cornwall29 tested 90 antibodies (specificities not given) and found that 63 showed reduced titration scores, and 25 were nonreactive when testing RBCs that had been treated with CDP for 30 minutes at 37°C. Most of these 25 antibodies were low titer or low avidity. They also treated RBCs with CDP at room temperature and saw a loss of reactivity, although less than that at 37°C.29 These investigators thus cautioned the use of rare single donor antibodies that are “low titer” or “low avidity” to obtain a phenotype on CDP-treated RBCs because of possible antigen weakening.29 Factors affecting the strength of antigens on CDP-treated RBCs include time and temperature of treatment; concentration, volume, and pH of CDP solution; and potency of antisera. Weaker results with treated RBCs will be more apparent if titration scores are used for comparison rather than results with undiluted antisera.

CDP does not always completely remove IgG from RBCs. Different publications show a wide variation (27–96%) in the effectiveness of CDP (Table 1). The variations may be due to differences in the antibody-coated RBCs and the CDP treatment methods used. RBCs with only partial removal of IgG may still be useful for antigen typing or autoadsorption purposes. Most studies comparing CDP with other methods found more effective IgG removal using EDTA acid (Table 1).

Another disadvantage of CDP, compared with EDTA acid, is that CDP treatment requires longer incubation time. At room temperature, CDP treatment requires a minimum incubation of 30 minutes (maximum, 2 hours) whereas EDTA acid treatment requires no more than a 2-minute incubation. In urgent situations where antigen typing must be performed on DAT+ RBCs—for example, to confirm the specificity of alloantibodies—it may be more practical to use EDTA acid than CDP. CDP, like EDTA acid, does not dissociate membrane-bound complement, so it is important to use anti-IgG when testing CDP-treated or EDTA acid-treated RBCs. Note that, unlike CDP or EDTA acid, ZZAP can be used to remove RBC-bound complement.30

Use of CDP


When using commercially manufactured CDP for clinical testing, it is necessary to follow the manufacturer’s recommendations. Any steps taken outside the manufacturer’s recommendation should be validated in-house before implementing their use. If there is a concern about weakening of antigen by CDP treatment, an aliquot of RBCs known to carry a single dose expression of the antigen(s) under investigation should be treated and tested in parallel. The CDP-treated RBCs should be tested with a protein medium (e.g., 6–10% albumin) in parallel with the blood grouping reagents. If the results with the albumin control are positive, then the antigen typing results are invalid.

References

1. Rodberg K, Tsuneta R, Garratty G. Discrepant Rh phenotyping results when testing IgG-sensitized RBCs with monoclonal Rh reagents (abstract). Transfusion 1995;35(Suppl):67S.

2. Lee E, Hart K, Burgess G, et al. Efficacy of murine monoclonal antibodies in RBC phenotyping of DAT-positive samples. Immunohematology 2006;22:161–5.

3. DePalma H, Lomas-Francis C, Wilson-Sandberg K, et al. Unexpected antigen typing discrepancies in testing of DAT+ RBCs with monoclonal reagents (abstract). Transfusion 2017;57(Suppl S3):37A.

4. Holtz G, Mantel W, Buck W. The inhibition of antigen-antibody reactions by chloroquine and its mechanism of action. Z Immunitatsforsch Exp Klin Immunol 1973;146:145–57.

5. Mantel W, Holtz G. Characterisation of autoantibodies to erythrocytes in autoimmune haemolytic anaemia by chloroquine. Vox Sang 1976;30:453–63.

6. Edwards JM, Moulds JJ, Judd WJ. Chloroquine dissociation of antigen-antibody complexes: a new technique for typing red cells with a positive antiglobulin test. Transfusion 1982;22: 59–61.

7. Widmann FK, Ed. AABB technical manual. 8th ed. Philadelphia, PA: JB Lippincott, 1981.

8. Branch DR, Petz LD. A new reagent (ZZAP) having multiple applications in immunohematology. Am J Clin Pathol 1982; 78:161–7.

9. Petz LD, Garratty G. Acquired immune hemolytic anemia. New York: Churchill Livingstone, 1980.

10. Louie JE, Jiang AF, Zaroulis CG. Preparation of intact antibody-free red blood cells in autoimmune hemolytic anemia (abstract). Transfusion 1986;26:550.

11. Kosanke J. EDTA glycine acid treatment of red blood cells. Immunohematology 2012;28:95–6.

12. McCullough JS, Torloni AS, Brecher ME, et al. Microwave dissociation of antigen-antibody complexes: a new elution technique to permit phenotyping of antibody-coated red cells. Transfusion 1993;33:725–9.

13. Swanson JL, Sastamoinen R. Chloroquine stripping of HLA A,B antigens from red cells (letter). Transfusion 1985;25: 439–40.

T. Aye and P.A. Arndt

14. Champagne K, Spruell P, Chen J, et al. EDTA/glycine-acid versus chloroquine diphosphate treatment for stripping Bg antigens from red blood cells. Immunohematology 1999; 15:66–8.

15. Gamma-Quin. Manufacturer’s instructions. Norcross, GA: Immucor, 2013.

16. Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific, 1998.

17. Burich MA, AuBuchon JP, Anderson HJ. Evaluation of rapid antibody dissociation techniques. Immunohematology 1986;2:76–80.

18. Burin des Roziers N, Squalli S. Removing IgG antibodies from intact red cells: comparison of acid and EDTA, heat and chloroquine elution methods. Transfusion 1997;37:497–501.

19. Sererat TS, Veidt D, Dutched A. A quick and simple method for phenotyping IgG-sensitized red blood cells. Immunohematology 2000;16:154–6.

20. Katharia R, Chaudhary RK. Removal of antibodies from red cells: comparison of three elution methods. Asian J Transfus Sci 2013;7:29–32.

21. Sassetti R, Nichols D. Decreased antigenic reactivity caused by chloroquine (letter). Transfusion 1982;22:537. Moulds JJ, Edwards JM, Judd WJ (response). Transfusion 1982;22: 537–8.

22. Prewitt PL, Steane EA, Steane SM. Decreased agglutinability with anti-D of chloroquine-treated red cells (abstract). Transfusion 1983;23:434.

23. Chen J, Spruell P, Moulds M. Studies using chloroquine diphosphate to strip HLA A and B antigens from red blood cells (RBCs). ISBT/AABB Book of Abstracts 1990;152.

24. Beaumont AE, Stamps R, Booker DJ, Sokol RJ. An improved method for removal of red cell-bound immunoglobulin using chloroquine solution. Immunohematology 1994;10:22–4.

25. Reid ME, Lomas-Francis C, Olsson ML. The blood group antigen factsbook. San Diego, CA; Academic Press, 2012.

26. Mallory D, Reid M. Misleading effects of chloroquine (letter). Transfusion 1984;24:412.

27. Shin C, Mallory D, Reid M. False negative red cell antigen typing following chloroquine treatment (abstract). Transfusion 1984;24:416.

28. Brazell J. Use of monoclonal Jk(a) and Jk(b) reagents in phenotyping red cells with a positive direct antiglobulin test. Immunohematology 1994;10:16–8.

29. McShane K, Cornwall S. Chloroquine reduces antigen strength (letter). Transfusion 1985;25:83.

30. Leger RM, Garratty G. A reminder that ZZAP reagent removes complement in addition to IgG from coated RBCs. Immunohematology 2006;22:205–6.

Thandar Aye, MLS(ASCP)SBB, Senior Technologist (corresponding author), Immunohematology Reference Laboratory, Bloodworks NW, 921 Terry Avenue, Seattle, WA 98104, [email protected]; and Patricia A. Arndt, MS, MT(ASCP)SBB, Lead Technologist, Special Immunohematology Laboratory, American Red Cross Blood Services, Southern California Region, Pomona, CA.


This article reviews information regarding the clinical significance of antibodies to antigens in the Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido/Rodgers, H, Kx, Cromer, Gerbich, Knops, Indian, and Ok blood group systems. Like most blood group systems, antibodies to many of the antigens in these groups are rarely encountered because of the high prevalence of the associated antigens in most populations. For many, the clinical significance—that is, the potential to cause reduced survival of transfused antigen-positive red blood cells or a transfusion reaction (e.g., anti-Ge2, anti-H) and/or hemolytic disease of the fetus and newborn (e.g., anti-Coa, anti-Ge3)—has been documented. Some of these antibodies are not always clinically significant, and because of the high prevalence of the antigen, antigen-negative blood may be extremely difficult to find (e.g., anti-LW, anti-Inb). The use of a monocyte monolayer assay may be helpful when making transfusion decisions for patients with these antibodies. For others, their prevalence is so rare that information on the clinical significance of their antibodies is not available (e.g., anti-Co4, anti-Ok). Immunohematology 2018;34:103–108.

Key Words: clinical significance, antibodies to red blood cell (RBC) antigens, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido/Rodgers, H, Kx, Cromer, Gerbich, Knops, Indian, Ok

Scianna Blood Group System

The Scianna (SC) blood group system, named in 1974, contains seven antigens: five high-prevalence antigens (Sc1, Sc3, Sc5, Sc6, and Sc7) and two low-prevalence antigens (Sc2 and Sc4) (Table 1).1 These antigens result from variants in the erythroid membrane-associated protein (ERMAP).2 The Sc protein is expressed weakly on leukocytes and other tissues such as fetal liver, thymus, lymph nodes, spleen, and bone marrow in adults.1

Clinical SignificanceSC antibodies are usually IgG and are detected by the

indirect antiglobulin test (IAT). There are rare cases of hemolytic disease with enough information to convincingly attribute clinical relevance to SC antibodies (Table 1).3 If possible, antigen-negative blood should be selected. SC:–1,2,3 blood may be available but is extremely rare. Donors being SC:–1,–2,–3 are not available.4

Antibodies to the two low-prevalence antigens, Sc2 and Sc4, have not been reported to cause hemolytic transfusion reactions (HTRs). However, they have been implicated in hemolytic disease of the fetus and newborn (HDFN). In the case of transfusion, IAT-compatible blood (most donors) should be selected.2


Clinical significance of antibodies to antigens in the Scianna, Dombrock, Colton, Landsteiner-Weiner, Chido/Rodgers, H, Kx, Cromer, Gerbich, Knops, Indian, and Ok blood group systemsS. Lejon Crottet

Revie w

Table 1. Antigen and antibody characteristics of the Scianna blood group system

Antigen Antibody

ISBT name Trivial name Prevalence HTR HDFN

SC1 Sc1 High No report No report (DAT+)

SC2 Sc2 Low No No to Mild (1 case) (DAT+)

SC3 Sc3 High No to mild/delayed

Mild

SC4 Rd Low No Mild to severe

SC5 STAR High No report No report

SC6 SCER High No report No report

SC7 SCAN High Yes, delayed (1 case)

No report

ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; DAT = direct antiglobulin test.


Dombrock Blood Group System

Besides the two antithetical antigens, Doa and Dob, the Dombrock blood group system (DO) contains eight antigens of high prevalence: Gya, Hy, Joa, DOYA, DOMR, DOLG, DOLC, and DODE (Table 2).1,5,6 Doa and Dob are polymorphic with a prevalence of 67 and 82 percent, respectively, in Caucasian individuals. DO is a glycoprotein attached to the red blood cell (RBC) membrane by a glycosylphosphatidylinositol anchor.5

Clinical SignificanceAnti-Doa and -Dob have caused immediate and delayed

HTRs, but no clinical HDFN (Table 2).1 They are mostly found in sera containing multiple RBC antibodies and often disappear in vivo. Anti-Doa and -Dob are usually IgG-reacting, optimally by the IAT using papain-treated RBCs.

For blood transfusion, antigen-negative blood should be selected.4 Because of the lack of reliable anti-Do antisera, it used to be difficult to find compatible blood. With today’s approach of molecular testing of donors, compatible donors are more easily found. Note, though, that the presence of other antibody specificities may complicate the finding of compatible blood.

Anti-Gya, -Hy, -Joa, and other DO antibodies are rare but have been reported to cause HTRs. Because of the rarity of these antibodies, evidence of clinical significance is limited

(Table 2).5 Serologically least-incompatible RBC units are recommended for patients with weak examples of the antibody, but antigen-negative RBCs should be provided for patients who demonstrate strong examples of the antibody.4

Colton Blood Group System

The Colton blood group system (CO) consists of three high-prevalence antigens (Coa, Co3, and Co4) and one polymorphic antigen (Cob) (Table 3). The water channel-forming protein, aquaporin-1, is the carrier of the CO blood group antigens.1

Clinical SignificanceAnti-Coa has caused delayed HTRs and severe HDFN,

and patients with anti-Coa should be transfused with Co(a–) blood.4

Whereas anti-Coa is often seen as a single specificity, anti-Cob is not. This rare antibody, detecting an antigen with a prevalence of about 10 percent,1 has been reported to cause both acute HTR and a mild delayed HTR (Table 3).7 About 90 percent of the donors are IAT compatible; these donor RBC units should be selected in case of a transfusion. There are no reports of anti-Cob being implicated in a serious HDFN, although mild HDFN has been reported.1

Anti-Co3 and anti-Co4 are rare antibodies detecting antigens of very high prevalence (Table 3). Anti-Co3 has caused a mild HTR and serious HDFN. Only four Co4– probands have been identified, and because of the rarity of anti-Co4, no data are available on its clinical significance.1 Co(a–b–) blood should be selected for compatibility testing, but because it is extremely rare serologically, least incompatible blood may be given with extra caution.4

Table 2. Antigen and antibody characteristics of the Dombrock blood group system

Antigen Antibody


DO1 Doa Polymorphic Yes; I/D No report (DAT+)

DO2 Dob Polymorphic Yes; I/D No report (DAT+)

DO3 Gya High No to moderate; D

No report (DAT+)

DO4 Hy High No to moderate; D

No report (DAT+)

DO5 Joa High No to moderate; D

No report

DO6 DOYA High No report (one case,

pretransfusion medication)

No report

DO7 DOMR High No report No report

DO8 DOLG High No report No report

DO9 DOLC High No report No report

DO10 DODE High No report No report

ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; I = immediate; D = delayed; DAT = direct antiglobulin test.

Table 3. Antigen and antibody characteristics of the Colton blood group system

Antigen Antibody


CO1 Coa High No to moderate/ D or I/H

Mild to severe

CO2 Cob Polymorphic No to moderate/D/H

Mild

CO3 Co3 High Mild; H Severe

CO4 Co4 High No report No report

ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; D = delayed; I = immediate; H = hemolytic.

S. Lejon Crottet


Landsteiner-Wiener Blood Group System

Currently, there are three antigens assigned to the Landsteiner-Wiener blood group system (LW): LWa, LWab, and LWb (Table 4).1 The LW antigens are carried on an intracellular adhesion molecule, glycoprotein ICAM-4.7 LWa and LWb are antithetical, with LWa being of high prevalence (100%) and LWb being of low prevalence (<1%) in most populations. The prevalence of LWb is greatest in the Baltic region (e.g., 8% in Estonians, 6% in Finns), and thus the likelihood of finding LW(a−) donors is greatest in these areas.1

Autoantibodies developed after antigen suppression are not uncommon, which makes the differentiation between allo- and autoantibodies difficult.

Clinical SignificanceLW antibodies are usually IgG and are detected by the

IAT. Mild cases of HTR and HDFN with anti-LWa, anti-LWab, and anti-LWb have been reported (Table 4).1 For transfusion, antigen-negative blood is not required, but D– blood should be selected if possible, since the antigen expression is lower than that of D+ blood. In the presence of anti-LWb, IAT-compatible blood should be selected.4

Chido/Rodgers Blood Group System

The antigens of the Chido/Rodgers blood group system (CH/RG) are carried on the C4d fragment of complement component C4; Ch antigens are carried on the C4B allotype and Rg antigens are carried on the C4A allotype.1 These antigens adsorb onto the RBC surface in vivo.1,8 Currently, nine antigens have been identified in the CH/RG system: six of high prevalence and one that is polymorphic for CH and two of high prevalence for RG (Table 5).

Clinical SignificanceAnti-Ch and anti-Rg have not been found to cause an HTR

or HDFN (Table 5).2 Antigen-negative blood is not required for transfusion. Severe anaphylactic reactions have been reported in a few patients with antibodies to Ch or Rg antigens after infusion of plasma products and platelet concentrates since these contain soluble Ch/Rg antigen; however; these events are exceptional.8

H Blood Group System

The H blood group system contains only one antigen, H. The H antigen is present in all individuals except those with the Bombay (Oh) phenotype. Individuals with the para-Bombay (Ah or Bh) phenotype have very low levels of H (Table 6).1

Clinical SignificanceIndividuals with the Oh phenotype (RBC H-deficient,

non-secretor) always have anti-H in their serum/plasma. Like anti-A and -B, anti-H is likely to cause a severe immediate HTR. HDFN may occur, but there are no reports (Table 6).1 In case of transfusion, blood of the Oh phenotype must be selected.

Individuals with the non-secretor Ah or Bh phenotype usually have anti-H in their serum/plasma, although often of lower titer. There is little information on the clinical


Table 4. Antigen and antibody characteristics of the Landsteiner-Wiener blood group system

Antigen Antibody


LW5 LWa High No to mild; D No to mild

LW6 LWab High No report Mild

LW7 LWb Low No to mild No to mild

ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; D = delayed.

Table 5. Antigen and antibody characteristics of the CH/RG blood group system

Antigen Antibody


CH/RG1 Ch1 High No hemolytic No report

CH/RG2 Ch2 High No report No report

CH/RG11 Rg1 High No hemolytic No report

CH/RG12 Rg2 High No report No report

Five additional antigens within the CH/RG blood group system have been identified but with no information.1

ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn.

Table 6. Antigen and antibody characteristics of the H blood group system

Antigen Antibody


H H High No to severe; I/D/H

No report

ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; I = immediate; D = delayed; H = hemolytic.


significance of anti-H in Ah or Bh individuals. Preferably, blood of the Oh phenotype should be transfused, but if not available, then RBCs of the appropriate ABO group (A for Ah, B for Bh) may be transfused with extra caution.4

Individuals with the secretor Ah or Bh phenotype may have anti-HI present in their serum/plasma. Typically, anti-HI is not reactive at 37°C, and ABO-identical blood, compatible at 37°C, can be used for transfusion.4

Anti-HI may also be found in group A1, A1B, or B individuals. If the antibody is reactive at 37°C, blood of the ABO group of the patient should be used for transfusion; blood of group O and A2 should not be used. If the antibody is reactive only at lower temperatures, blood compatible at 37°C can be used for transfusion, regardless of ABO group.4

Kx Blood Group System

Kx was assigned blood group system status in 1990 and contains one antigen of very high prevalence (100%), Kx (Table 7). The Kx antigen is covalently linked to the Kell molecule on RBCs and is controlled by a gene on the X chromosome. The presence of the Kx protein is critical to normal RBC morphology, and silencing mutations are associated with McLeod syndrome, neuroacanthocytosis, and neuromuscular disorders in later life.9

Clinical SignificanceAnti-Kx is often found together with anti-Km (anti-

KEL20) in men with the McLeod phenotype and X-linked chronic granulomatous disease. These two antibodies (also called anti-KL) can cause severe HTRs; thus, antigen-negative (McLeod phenotype) blood should be selected for transfusion (Table 7).4

Gerbich Blood Group System

The Gerbich blood group system is expressed on glycophorin C and D and contains 11 antigens: 6 of high prevalence and 5 of low prevalence (Table 8).10

Clinical SignificanceAnti-Ge2, made by Yus, Gerbich, or Leach phenotypes,

and anti-Ge3, made by Gerbich or Leach phenotypes, are usually IgG antibodies and can cause moderate immediate or delayed HTRs. Anti-Ge4 (made by Leach phenotypes), -GEPL, -GEAT, and -GETI are very rare, and no data are available on their clinical significance (Table 8).10 Antigen-negative blood is not usually required for transfusion but should be considered for strong examples of these antibodies.4

Although Anti-Ge2 has not been implicated in clinical HDFN, newborns have shown positive direct antiglobulin tests (DATs).1 Anti-Ge3 has caused severe HDFN due to suppression of the erythroid progenitor cell growth in the infant. Initial treatment of the infant at birth with subsequent follow-up for several weeks after birth may be indicated.1 There is no information on the clinical significance with regard to HDFN for anti-Ge4, -GEPL, -GEAT, and -GETI.1

The antibodies to the low-prevalence antigens of the Gerbich system can be naturally occurring and may be IgM

S. Lejon Crottet

Table 7. Antigen and antibody characteristics of the Kx blood group system

Antigen Antibody


XK1 Kx High Mild; D Not applicable*

*Anti-Kx only made by male individuals with McLeod phenotypeISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; D = delayed.

Table 8. Antigen and antibody characteristics of the Gerbich blood group system

Antigen Antibody


GE2 Ge2 High No to moderate; I/D

No report (DAT+)

GE3 Ge3 High No to moderate; I/D

Positive DAT to severe

GE4 Ge4 High No report No report

GE5 Wb Low No report No report

GE6 Lsa Low No report No report

GE7 Ana Low No report No report

GE8 Dha Low No report 1 report, very severe

GE9 GEIS Low No report No report

GE10 GEPL High No report No report

GE11 GEAT High No report No report

GE12 GETI High No report No report

ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; I = immediate; D = delayed; DAT = direct antiglobulin test.



or IgG. There are no reports that these antibodies have caused HTRs or HDFN.1

Cromer Blood Group System

The antigens of the Cromer blood group system are carried on the decay accelerating factor (DAF), a protein belonging to the regulators of the complement activation family. The system contains 16 high-prevalence antigens and three low-prevalence antigens (Table 9).11

Clinical SignificanceAntibodies to Cromer antigens are rare (Table 9).

There is evidence that the antibodies may cause accelerated destruction of transfused RBCs, although functional assays on clinical significance are ambiguous.11 Because of the high concentration of maternal DAF on the apical surface of the trophoblasts in the placenta, which is thought to adsorb the maternal antibodies, there is no risk of HDFN associated with Cromer system antibodies. Antigen-negative blood is not usually required for transfusion, but should be considered for strong-reacting antibodies.4

Knops Blood Group System

The antigens of the Knops blood group system are carried on complement receptor 1 (CR1). The major functions of CR1 are the binding of C4b/C3b opsonized complexes as well as complement regulation. The Knops blood group system contains nine antigens that are polymorphic, of low prevalence or of high prevalence depending on the origin of the individual (Table 10).1,12

Clinical SignificanceMost of the Knops antibodies are not considered clinically

significant since they do not cause apparent HTRs or HDFN, thus antigen-negative units are not indicated in the case of transfusion (Table 10).4

Indian Blood Group System

The Indian blood group system contains four antigens that are carried by CD44. Ina and Inb are antithetical antigens of low prevalence and high prevalence, respectively. INFI and INJA are both antigens of high prevalence (Table 11).2

Table 9. Antigen and antibody characteristics of the Cromer blood group system

Antigen Antibody

ISBT name Trivial name Prevalence HTR HDFN*

CROM1 Cra High No to moderate No

CROM2 Tca High No to severe No

CROM3 Tcb Low No report No report

CROM4 Tcc Low No to mild No

CROM5 Dra High No to mild No

CROM6 Esa High Mild No

CROM7 IFC High No to mild No

CROM8 WESa Low No to mild No

CROM9 WESb High No report No, DAT+

CROM10 UMC High No report No

CROM11 GUTI High No report No

CROM12 SERF High No data No

CROM13 ZENA High No data No

CROM14 CROV High No data No

CROM15 CRAM High No data No

CROM16 CROZ High No data No

*Decay accelerating factor adsorbs maternal antibodyISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; DAT = direct antiglobulin test.

Table 10. Antigen and antibody characteristics of the Knops blood group system

Antigen Antibody


KN1 Kna High No No

KN2 Knb Low No report No report

KN3 McCa High No No

KN4 Sla High (non-black)

Polymorphic (black)

No to mild No

KN5 Yka High No No

KN6 McCb Low (non-black)

Polymorphic (black)

No report, unlikely

No report

KN7 Vil Low (non-black)

Polymorphic (black)

No report, unlikely

No report

KN8 Sl3 High No report, unlikely

No report

KN9 KCAM High (non-black)

Polymorphic (black)

No report, unlikely

No report



Clinical SignificanceIndian antibodies are mostly IgG, reacting preferably by

the IAT (Table 11). These antibodies do not bind complement and do not cause in vitro hemolysis.6 Ina is rare in populations of European origin, but has a prevalence of about 3 percent in Indian individuals and 10 percent in Arab individuals. Anti-Ina is not reported to be clinically significant, although limited data are available. Blood compatible by IAT can be selected for transfusion.4

Anti-Inb may cause none to severe/delayed HTRs.1 In(b–) blood is not usually required for transfusion but should be considered for strong-reacting antibodies.

No clinical data are available on anti-INFI and anti-INJA; INFI– and INJA– blood types are extremely rare. For transfusion, serologically least-incompatible blood should be used with extra caution.4

Anti-Ina, -Inb, and -INJA have not been implicated in HDFN.1 Anti-INFI was implicated in one case of mild HDFN.13

Ok Blood Group System

The Ok blood group system contains three antigens carried on CD147 (Table 12).14 The Ok blood group system was established in 1999. To date, the null phenotype, Ok(a–) has been found in eight Japanese families only.

Clinical SignificanceOk antibodies have not been implicated in HDFN.1 There

is almost no information on the clinical significance of Ok antibodies, but in vivo survival tests and cellular functional assays suggest that anti-Oka is clinically significant (Table 12).1 Because Ok(a–) blood is extremely rare, serologically least-incompatible blood may be given with extra caution.4

References

1. Reid ME, Lomas-Francis C, Olsson ML. Blood group antigen factsbook. 3rd ed. San Diego, CA: Academic Press, 2012.

2. Wagner FF, Poole J, Flegel WA. Scianna antigens including Rd are expressed by ERMAP. Blood 2003;101:752–7.

3. Bunker PAR, Flegel WA. Scianna: the lucky 13th blood group system. Immunohematology 2011;27:41–57.

4. Thornton N. The clinical significance of blood group alloantibodies and the supply of blood for transfusion, 2016. SPECIFICATION SPN214/4. Available from http://hospital.blood.co.uk/media/29271/spn2144-the-clinical-significance-of-blood-group-alloantibodies-and-the-supply-of-blood-for-transfusion.pdf.

5. Lomas-Francis C, Reid ME. The Dombrock blood group system: a review. Immunohematology 2010;26:71–8.

6. Storry JR, Castilho L, Chen Q, et al. International society of blood transfusion working party on red cell immunogenetics and terminology: report of the Seoul and London meetings. ISBT Sci Ser 2016;11:118–22.

7. Bryne KM, Bryne PC. Review: other blood group systems—Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, and Indian. Immunohematology 2004;20:50–8.

8. Daniels G. Human blood groups. 3rd ed. Oxford, U.K.: Blackwell Science, 2013.

9. Denomme GA. Kell and Kx blood group systems. Immunohematology 2015;31:14–9.

10. Walker PS, Reid ME. The Gerbich blood group system: a review. Immunohematology 2010;26:60–5.

11. Storry JR, Reid ME, Yaser MH. The Cromer blood group system: a review. Immunohematology 2010;26:109–18.

12. Moulds JM. The Knops blood group system: a review. Immunohematology 2010;26:2–7.

13. Poole J, Tilley L, Warke N, et al. Two missense mutations in the CD44 gene encode two new antigens of the Indian blood group system. Transfusion 2007;47:1306–11.

14. Smart EA, Storry JR. The OK blood group system: a review. Immunohematology 2010;26:124–6.

Sofia Lejon Crottet, PhD, Head of Immunohematology, Interregional Blood Transfusion Service, Swiss Red Cross, Bern, Switzerland, [email protected].

S. Lejon Crottet

Table 11. Antigen and antibody characteristics of the Indian blood group system

Antigen Antibody


IN1 Ina Low Decreased cell survival

No, DAT+

IN2 Inb High No to severe D No, DAT+

IN3 INFI High No report Mild

IN4 INJA High No report No report

ISBT = International Society of Blood Transfusion; HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; DAT = direct antiglobulin test; D = delayed.

Table 12. Antigen and antibody characteristics of the Ok blood group system

Antigen Antibody


OK1 Oka High 51Cr cell survival reduced

No

OK2 OKGV High No report No report

OK3 OKVM High No report No report



The Rh system is the most complex of the human blood groups. Of the 55 antigens that have been characterized, the system’s principal antigens D, C, E, c, and e are responsible for the majority of clinically significant Rh antibodies. In the last few years, advancements in molecular testing have provided a wealth of information on the genetic diversity of the Rh locus. This case report describes a patient with variant RHD*DAR alleles inherited in conjunction with two compound heterozygote RHCE*ceEK/RHCE*ceAR alleles. The patient was previously alloimmunized to D, C, and E and possibly hrS. Further transfusion of D–C–E–K– RBCs resulted in a suspected acute hemolytic transfusion reaction and the subsequent identification of anti-c. Monocyte monolayer assay testing suggests clinical significance with a range of 29.5–38.5 percent reactive monocytes. Immunohematology 2018;34:109-112.

Key Words: RH variant alleles, anti-hrS, RH allele matching, partial antigens, Rh antigens, delayed hemolytic transfusion reaction, monocyte monolayer assay, hrS

The Rh system is the most complex of the human blood groups. Of the 55 antigens that have been characterized, the system’s principal antigens D, C, E, c, and e are responsible for the majority of clinically significant Rh antibodies. Antibodies to antigens in the Rh system have been implicated in hemolytic disease of the fetus and newborn (HDFN) as well as acute and delayed hemolytic transfusion reactions (HTRs). In the last few years, advancements in molecular testing have provided a wealth of information on the genetic diversity of the Rh locus. The Rh system is a very polymorphic and immunogenic blood group system that is second only to the ABO system in its clinical significance for blood transfusion. Nucleotide changes can result in qualitative and quantitative changes in Rh antigen expression.1 Individuals who express a partial D on their red blood cells (RBCs) can be immunized to make anti-D. Similarly, people who are homozygous or hemizygous for alleles encoding partial c and/or partial e can also produce alloantibodies.2 In addition to the common antigens of the Rh system (D, C, c, E, e) there are 50 additional antigens that constitute the Rh blood group system.3 The Rh

locus consists of a pair of adjacent homologous genes, RHD and RHCE, that respectively encode for D and for C or c and E or e antithetical pairs. The antigenic diversity present in this blood group system is attributed to a variety of molecular mechanisms including nucleotide substitutions and gene conversion.4 These molecular mechanisms lead to the creation of unique protein sequences with resultant unique antigens and an increase in the number of antigens in the Rh system relative to other blood group systems. Further adding to the complexity, variant RHD and RHCE alleles can encode qualitative and/or quantitative changes in the Rh antigen expression.5 Individuals with such variants, when exposed to the conventional antigen during transfusion, will recognize those epitopes that they lack as foreign, and an immune response may be elicited. Routine serologic RBC phenotyping does not differentiate between conventional and variant Rh antigens. Within the Rh blood group system, partial D, C, and e variants and their corresponding alloantibodies have been well described. Alloanti-c in a c+ individual was first reported by Moulds et al.6 in 1982, with a number of additional cases reported since that time.7–9

Peyrard et al.10 reported the first case of alloanti-c/ce in a patient with the RHCE*ceAR allele that encodes a partial c antigen. Hipsky et al.11 also described the identification of alloanti-c in the plasma of an African American patient with sickle cell disease carrying the RHCE*ceAR allele.

We report a case of a 72-year-old multi-transfused African American woman with altered RHD and RHCE alleles predicting partial D, partial c, and partial e antigens who developed a clinically significant alloanti-c.

Case Report

The patient is a 72-year-old African American woman with a past medical history of multiple sclerosis, congestive heart failure, and recurrent gastrointestinal (GI) bleeding secondary to diverticulosis. The patient presented to the

A delayed and acute hemolytic transfusion reaction mediated by anti-c in a patient with variant RH allelesT.K. Walters and T. Lightfoot

Case RepORt


Table 1. Molecular testing results

Allele name Antigens encoded by allele

Probable RHD genotype

RHD*DAR (hemizygous or homozygous)

Partial D

Probable RHCE genotype

RHCE*ceEK/ Partial c, partial e, Hr–, hrS–

RHCE*ceAR Partial c, partial e, Hr–, hrS–, V+w, VS–

Predicted phenotype

Partial D+C–E– partial c+ partial e+ Hr– V+w VS– hrS–

emergency department with acute GI bleeding and received fluid support and 2 units of packed red blood cells (PRBCs). She was admitted to the hospital for further evaluation and treatment. A colonoscopy was performed that revealed a diverticular bleed in the ascending colon; this bleed was successfully cauterized. During this time, her hemoglobin (Hgb) was observed to fall to 4.9 g/dL with a hematocrit (Hct) of 16 percent (normal 12.0–15.5 g/dL and 37–48%, respectively); she was emergently transfused 4 units of PRBCs. Initial serologic results indicated the patient to be group A, weak D positive with a positive antibody detection test. The patient’s previous serologic history included identification of anti-D, -C, -E, warm autoantibody, and cold autoantibody. The initial antibody screening was performed by the hospital, and the presence of additional RBC antibodies was suspected. The patient’s sample was referred to the Immunohematology Reference Laboratory (IRL) (American Red Cross, Charlotte, NC) for further serologic evaluation and antibody identification.

Results

Initial serologic testing performed by the IRL indicated that the patient was group A, weak D positive with a negative direct antiglobulin test (DAT). Serologic phenotype testing was performed and the results were C–E–c+e+; K–; Fy(a–b–); Jk(a+b+); S+s–. The patient’s plasma exhibited panreactivity with D– phenotypically similar RBCs in the presence of a negative autocontrol. This reactivity suggested the presence of an additional antibody (ies) unrelated to the previously identified alloanti-D, -C, and -E. Plasma studies showed 1+ reactivity with D– phenotypically similar cells using low-ionic-strength saline solution in the indirect antiglobulin test (IAT) and 3+ reactivity with polyethylene glycol (PEG) in the IAT. The reactivity was resistant to ficin treatment as well as to 0.2 M dithiothreitol. Given the patient’s history of warm and cold autoantibodies, adsorption studies were performed at 37°C using ZZAP-treated autologous cells. No reduction in strength was observed with the autoadsorbed plasma. Allogeneic adsorptions were performed using papain-treated R0 RBCs. Clinically significant antibodies to common RBC blood group antigens were excluded in the alloadsorbed plasma. An acid eluate was prepared from the adsorbing cells to ascertain the identity of the panreactivity. The acid eluate demonstrated an anti-e–like antibody. The eluate was reactive with three sources of D– e+ RBCs and negative with three sources of D– hrB– RBCs.

The facility was contacted to report the anti-e–like reactivity, and molecular genotype testing was recommended for further characterization. No compatible units negative for D, C, E, e, and K were available for immediate transfusion. Therefore, after consultation with the facility, units negative for D, C, E, and K were recommended in the event emergent transfusion was needed.

Unfortunately, there was insufficient time to perform a monocyte monolayer assay (MMA) before transfusion of incompatible blood.12 An MMA can be performed to help assess the clinical significance of an antibody when antigen-negative units are rare and not likely to be readily available. Given the patient’s clinical situation (GI bleed with a low Hgb level), the facility transfused 2 crossmatch-incompatible units of PRBCs negative for D, C, E, and K over a 48-hour time period. No adverse reactions were noted after transfusion of e+ units in the presence of the anti-e–like antibody.

Molecular testing was performed by the American Red Cross National Molecular Laboratory. Results indicated the patient carried a RHD*DAR variant allele inherited with compound heterozygote RHCE*ceEK/RHCE*ceAR alleles, predicting partial D, partial c, partial e, and the lack of the high-prevalence antigens, hrS and Hr (Table 1).

The molecular results confirmed suspected serologic reactivity. RHD*DAR allele is associated with D typing discrepancies and the production of alloanti-D. In addition, the patient had two altered RHCE alleles predicting the hrS– phenotype and the production of alloanti-e and/or anti-hrS. The facility was contacted to report the additional probable anti-hrS reactivity in the presence of previously identified anti-D, -C, and -E. Consultation was made with the American Rare Donor Program, and no RH allele–matched units were available for further transfusion needs. Despite the presence of the probable anti-hrS, the patient tolerated the previous 2 units of PRBCs well, with no adverse effects. The patient’s Hgb

T.K. Walters and T. Lightfoot


HTR due to anti-c

increased to 7.7 g/dL with an Hct of 23 percent, and she was discharged.

Five days after the initial transfusion, the patient was readmitted to the hospital for a suspected delayed HTR. The patient presented with dark urine, Hgb of 6.3 g/dL, total bilirubin of 2.6 mg/dL (normal 0.1–1.2 mg/dL), lactate dehydrogenase 1034 IU (normal 100–190 IU), and haptoglobin less than 15 mg/dL (normal 30–200 mg/dL). The patient then received 2 additional units of D–C–E–K– PRBCs.

On completion of transfusion of the second unit, the patient began to display symptoms of a possible acute HTR. The patient’s temperature increased from 98.0 to 99.5°F, with accompanying symptoms of rigors, dyspnea, and severe back pain. Blood pressure increased from 149/68 mmHg to 166/94 mmHg, with an increase in pulse from 85 bpm to 93 bpm. Oxygen saturation remained unchanged at 99 percent.

The facility began an initial transfusion reaction investigation. The post-transfusion sample was severely hemolyzed, but the DAT remained negative. Gram stain was performed and was negative for any microorganisms. A current blood sample was drawn and sent to the local IRL for additional serologic testing. The DAT on both the pre- and post-transfusion samples was negative.

Because of the recent transfusion and suspected transfusion reaction, an eluate was prepared despite the negative DAT. Expecting the eluate to contain the previously identified probable anti-hrS, the eluate was tested against a panel of e+ and e– cells. All cells were reactive 4+ at PEG-IAT, and the patient’s EDTA glycine acid–treated autologous cells were negative, as was the last wash. All cells tested with the eluate were positive except for Rhnull RBCs. Two Rhnull RBCs were tested and found to be negative with the patient’s neat eluate. Additionally, one RBC with the Rh:–46 phenotype was available for testing and was significantly weaker in reactivity and displayed only a 1+ reaction. This cell was negative for c and had a weakened expression of e. Given that the patient’s molecular results indicated an altered expression of c, the possibility of anti-c or anti-Hr was suspected. The eluate was tested against a panel of hrS– cells to investigate the presence of anti-Hr, but all cells were 3+ reactive with the neat eluate. The eluate was adsorbed using R1R1 cells. Anti-c was demonstrated in the adsorbed eluate at PEG-IAT. Additionally, the previously identified anti-hrS was also shown to be demonstrating in the neat eluate when tested with a c–e+ RBC.

Plasma studies were also performed on the post-transfusion sample. The plasma was reactive 4+ with two phenotypically similar cells [D–C–E–s–K–Fy(a–b–)]. The

autocontrol remained negative. Adsorption studies were performed using aliquots of R1R1 cells. The adsorbed plasma demonstrated the presence of anti-c. To further confirm the anti-c specificity, an additional aliquot of the plasma was adsorbed with R2R2 cells. An acid eluate was then prepared from the adsorption cells. This eluate reacted with D–c+E– RBCs and was nonreactive with Rhnull RBCs. Clinically significant antibodies to other major blood group antigens were excluded in the allogeneic adsorbed eluate and plasma. The presence of anti-c in conjunction with anti-D, -C, -E, and probable anti-hrS presented significant challenges in the possibility of locating any compatible blood for this patient. An MMA was performed by the American Red Cross National Reference Laboratory for Blood Group Serology in an attempt to assess the clinical significance of the alloantibody to the c antigen. Two random sources of group O, D–C–E–K– RBCs and autogeneic cells were used. Results are listed in Table 2. Because the patient had antibodies to both c and e as well as to D, C, and E, phenotypically similar cells (D–C–E–) were chosen for the MMA. Cells that could further distinguish the significance of the reactivity with regard to the partial anti-c or probable anti-hrS were not available. MMA testing using genotypically similar RBCs would have been ideal, but none are currently available in the United States.

Discussion

We report the serologic and molecular findings on a 72-year-old multi-transfused African American female patient. The presence of anti-c in conjunction with anti-D, -C, -E, and probable anti-hrS posed significant challenges in the identification and provision of compatible blood for this patient. The MMA results, the patient’s clinical status, and post-transfusion laboratory and serologic results indicate that the alloanti-c is clinically significant.

Table 2. Monocyte monolayer assay results

IgG DAT* % Reactive monocytes†

Random #1 4+ 30.3 with fresh complement

D–C–E–K–Fy(a–b–)s– 4+ 34.0 without fresh complement

Random #2 4+ 38.5 with fresh complement

D–C–E–K– 4+ 29.5 without fresh complement

Autologous cells Negative 0.3 without fresh complement

*Results of coated test red blood cells after 37°C incubation.†The normal range for the monocyte monolayer assay is 0–3 percent reactive monocytes. Values above 3 percent suggest that the antibody may cause accelerated clearance of antigen-positive red blood cells.18

DAT = direct antiglobulin test.


T.K. Walters and T. Lightfoot

Variants of c are much less frequent than variant forms of the other major Rh antigens.12 Westhoff et al.13 postulate that the less frequent occurrence of c variants in comparison with other Rh antigens is because the two proline residues involved in c expression form a stable structure that is resistant to the changes that often occur with RHCE. Individuals of African descent have a much higher incidence of variant Rh antigens as compared with individuals of European descent, with the latter constituting the largest percentage of blood donors by ethnicity.14,15 The high prevalence of variant antigens in African American patients thereby increases the risk of alloimmunization when these patients are exposed to the conventional antigen during transfusion.16,17

The detection of the presence of a variant antigen, identification of the corresponding alloantibody, determination of its clinical significance, and the logistics of finding compatible blood can be quite challenging. Rhnull RBCs were recommended for any future transfusions for this patient, until such time that a possible RH allele–matched donor unit could be identified for further compatibility testing and evaluation. This case demonstrates the increasingly important role of molecular testing in the overall clinical/laboratory assessment of alloimmunized patients with partial RBC antigens and for subsequent transfusion recommendations and guidance.

Acknowledgments

The authors would like to thank Dr. Carol Weida (Atrium Health, Charlotte, NC) for assistance with the management of this case and the procurement of the clinical data for this manuscript. The authors would also like to thank the staff at the American Red Cross National Molecular Laboratory and National Reference Laboratory for Blood Group Serology for their assistance with molecular and MMA testing.

References

1. Fung MK, Eder AF, Spitalnik SL, Westhoff CM. Technical manual. 19th ed. Bethesda, MD: American Association of Blood Banks, 2017.

2. Reid ME, Lomas-Francis C, Olsson ML. The blood group antigens factsbook. 3rd ed. San Diego, CA: Elsevier, 2012.

3. International Society of Blood Transfusion. Table of blood group antigens v.8_180620. Available at www.isbtweb.org/fileadmin/user_upload/Red_Cell_Terminology_and_Immunogenetics/Table_of_blood_group_antigens_within_systems_v8_180620.pdf. Accessed August 2018.

4. Klein HC, Anstee DJ. Mollison’s blood transfusion in clinical medicine. 11th edition. Malden, MA: Blackwell Publishing, 2005.

5. Avent ND, Reid ME. The Rh blood group system: a review. Blood 2000;95:375–87.

6. Moulds JJ, Case J, Anderson TD, Cooper ES. The first example of allo-anti-c produced by a c-positive individual. In: Recent advances in haematology, immunology and blood transfusion: proceedings of the plenary sessions of the joint meeting of the 19th congress of the International Society of Haematology and the 17th Congress of the International Society of Blood Transfusion, Budapest, August 1982. Wiley, 1983.

7. Ong J, Walker PS, Schmulbach E, et al. Alloanti-c in a c-positive, JAL-positive patient. Vox Sang 2008;96:240–3.

8. Pham BN, Peyrard T, Juszczak G, et al. Alloanti-c (RH4) revealing that the (C)ces haplotype encodes a partial c antigen. Transfusion 2009;49:1320–4.

9. Hipsky CH, Lomas-Francis C, Fuchisawa A, et al. RHCE*ceCF encodes partial c and partial e not CELO, an antigen antithetical to Crawford. Transfusion 2011;51:25–31.

10. Peyrard T, Pham BN, Poupal S, et al. Alloanti-c/ce in a c+ ceAR/Ce patient suggests that the rare RHCE*ceAR allele (ceAR) encodes a partial c antigen. Transfusion 2009;49:2406–11.

11. Hipsky CH, Lomas-Francis C, Fuchisawa A, Reid M. RHCE*ceAR encodes a partial c (RH4) antigen. Immunohematology 2010;26:57–9.

12. Simon TL, McCullough J, Snyder EL, Solheim BG, Strauss RG. Rossi’s principles of transfusion medicine. 5th ed. West Sussex, UK: Blackwell Publishing, 2016.

13. Westhoff CM, Silberstein LE, Wylie DE. Evidence supporting the requirement for two proline residues for expression of c. Transfusion 1997;37:1123–30.

14. Wang D, Lane C, Quillen K. Prevalence of RhD variants, confirmed by molecular genotyping, in a multiethnic prenatal population. Am J Clin Pathol 2010;134:438–42.

15. Mezokh N, Song Y, Moulds JM, et al. Reliable identification of diverse RHD and RHCE variants in African American donors by Beadchip analysis (abstract). Transfusion 2009;49:120A.

16. Westhoff CM, Vege S, Horn T, et al. RHCE*ceMO is frequently in cis to RHD*DAU0 and encodes a hr(S)-, hr (B)-, RH:-61 phenotype in black persons: clinical significance. Transfusion 2013;53:2983–9.

17. Noizat-Pirenne F, Lee K, Pennec PY, et al. Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: Identification and transfusion safety. Blood 2002;100: 4223–31.

18. Nance SJ, Arndt P, Garratty G. Predicting the clinical significance of red cell alloantibodies using a monocyte monolayer assay. Transfusion 1987;27:449–52.

Tiffany K. Walters MT(ASCP)SBBCM, Director, (corresponding author), Immunohematology Reference Laboratory, American Red Cross, Carolinas Charlotte, Carolinas Durham, River Valley, Tennessee Valley, and South Carolinas Regions, 2425 Park Road, Charlotte, NC, 28203, [email protected]; and Thomas Lightfoot, MD, Medical Director, American Red Cross, Charlotte, NC, 28203.


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Polyagglutination is a condition in which red blood cells (RBCs) are agglutinated by normal adult human sera but not by autologous or newborn sera. Polyagglutination is caused by changes in the RBC membrane that enable patient RBCs to agglutinate with normal human sera; this agglutination can interfere with blood bank testing. Depending on the cause, polyagglutination may or may not be the cause of RBC hemolysis. Lectins and human sera can be used to detect polyagglutinable RBCs. Identification of this phenomenon can be helpful in providing not only transfusion recommendation information for physicians but also information associated with pathogens (i.e., Streptococcus pneumoniae) and severity of illness. Testing with ABO group–compatible adult human sera can determine if a patient’s RBCs are polyagglutinable. Further testing with a variey of lectins may identify the kind of polyagglutination. Immunohematology 2018;34:113–117.

Key Words: polyagglutination, lectin, acquired B, T, Tk, Th, Tx, Tr, HEMPAS, Cad, Tn

Principle and Background

Polyagglutination was first described in 1988 by Peter Hermann Stillmark in his doctoral thesis. The agglutinin used was isolated from seeds of the castor tree (i.e., ricin) and was highly toxic. Ricin became commercially available, which prompted Paul Ehrlich to use it for studies in the 1890s to establish fundamental principles of immunology. Ehrlich found that mice could become tolerant to a lethal dose of ricin by repeated small injections of the lectin, creating protective antibodies to the ricin.1

Karl Landsteiner detected hemagglutinating activity in some nontoxic plant extracts. In 1909, he observed that the hemagglutinating activity of certain seed extracts or “lectins” was inhibited by either heat-treated serum or by mucin.2 During the 1940s and 1950s, there was increased research in the area of lectin extracts from plant seeds. Some lectins showed hemagglutinating activity with all RBCs, and others showed blood group–specific activity, such as A or H.

There are two categories of polyagglutination: acquired (T, Tk, Th, Tx, acquired B) and inherited (Tr, HEMPAS, Cad, Tn). In both categories, the surface of human RBCs can become altered by (1) microbial enzyme modification, usually through sepsis; (2) homozygosity of rare altered alleles, often due to

consanguinity; and (3) a disease state such as lymphoma or leukemia. This RBC alteration causes panagglutination when these RBCs are combined with normal adult human sera which contain naturally occurring antibodies to these altered antigens but not when they are combined with cord sera which do not.3 Understanding the mechanisms by which polyagglutinable RBCs occur can help in recognizing these conditions when working on patient samples and in determining when polyagglutination screening is indicated.

Reagents/Supplies

Reagents Supplies

• Adult group O RBCs

• Phosphate-buffered saline (7.0–7.5 pH)

• Lectins (if available): Arachis hypogea, Glycine soja, Salvia sclarea, Salvia horminum (or Dolichos biflores if the patient is non–group A)

• Polyagglutinable RBCs (frozen or prepared)

• Donor pooled group AB plasma or serum

• Pooled cord serum

• Neuraminidase, 1 IU/mL from Vibrio cholera

• Centrifuge or cell washer

• Alsever’s solution

• 0.9% saline

• 6% albumin

• 10 × 75 mm tubes

RBCs = red blood cells.

Procedural Steps

Preparing controls• Treat adult group O RBCs with neuraminidase.• Pool the serum from three to five cord blood samples.• Grind up lectin seeds, incubate in 0.9% saline, and harvest the

supernatant.Process• Mix lectin and/or human sera with a 3–5% suspension of patient

RBCs.• Incubate at room temperature.• Centrifuge, and read macroscopically for agglutination.


Detecting polyagglutinable red blood cellsC. Melland and C. Hintz


Microbial Enzyme ModificationPolyagglutination caused by microbial enzyme

modification occurs when enzymes excreted from bacteria cleave portions of the RBC membrane, exposing hidden antigens termed “cryptantigens” on the RBC surface. There are many types of polyagglutination due to bacterial action including but not limited to T, acquired B, Tk, Th, and Tx. Identifying and understanding the type of polyagglutination often linked to pathogens can be helpful for the clinician.4,5

T-acTivaTion

During T-activation, the neuraminidase enzyme cleaves terminal N-acetylneuraminic acid (NeuAc) from the RBC membrane, exposing a hidden crypt or T antigen that can agglutinate with naturally occurring IgM anti-T present in most adult sera. This phenomenon was first described in 1938 when the RBCs of a 4-year-old child with bronchopneumonia were agglutinated by 15 percent of ABO-compatible human sera.6 Anti-T IgM antibodies are absent in newborns and form over time in response to T-like antigenic stimulation of bacteria from intestinal exposure. This process is analogous to the formation of anti-A and anti-B from exposure to A- and B-like antigens in the environment.7 Unlike anti-A and anti-B, however, the binding of anti-T has not been proven to cause hemolysis during the acute disease state. T-activated RBCs are agglutinated by human sera and by Arachis hypogea and Glycine soja.5,8

acquired B, Tk, Th, Tx Acquired B, Tk, Th, and Tx polyagglutination are

caused by similar processes. Acquired B is associated with gastrointestinal (GI) disease and occurs when A1 RBCs (N-acetylgalactosamine) are degraded by bacterial enzymes to galactosamine and therefore look similar to B RBCs but react with human anti-B.9

Before the advent of monoclonal reagents, human polyclonal reagents were used for routine ABO blood group testing. These reagents were prepared from human sera which contained naturally occurring antibodies to such “cryptantigens”. Acquired B polyagglutination caused blood group discrepancies in the forward typing results when polyclonal reagents were used.10 Tk polyagglutination is also associated with GI infection. Bacteria that produce endo-beta-galactosidase remove galactose residues, exposing N-acetylglucosamine in the paragloboside system and causing Tk polyagglutination.11 Th and Tx polyagglutination are not as well understood as the other types of microbial infection but

have been associated with bacterial infections of the intestinal tract and pneumococcal infections, respectively.12,13

Inheritance of Rare Altered AllelesPolyagglutination caused by the inheritance of rare, altered

alleles is associated with increased or altered glycosylation or changes in the antigen expression on the RBC surface, which causes the RBC to react with naturally occurring antibodies in human sera. Examples of inherited polyagglutination include but are not limited to Tr, HEMPAS, and Cad. NOR has now been assigned to the P1PK blood group system (ISBT 003.004 or 3.4) and is no longer considered a polyagglutinable state.14,15

Tr

Tr polyagglutination has been shown to be caused by the lack of glycosylation of membrane proteins.16 In this case, the parents were consanguineous, and the propositus was homozygous for the altered allele.

heMPaS HEMPAS is an acronym for hereditary erythroblastic

multinuclearity with a positive acidified serum lysis test and is caused by congenital dyserythropoietic anemia type II. The RBCs of these individuals react with anti-I and anti-i.17 These abnormal RBC membranes result in increased susceptibility to lysis and a compensating anemia.18 HEMPAS RBCs react with Glycine soja.

cad

Individuals with Cad polyagglutination do not have a compensating anemia but have a very strong expression of Sda on their RBCs which reacts with weak naturally occurring anti-Sda.19–21 Cad+ RBCs react with Glycine soja and Salvia horminum.

Polyagglutination and DiseasePolyagglutination has been associated with leukemia in

some individuals, and Tn polyagglutination characterized by persistent mixed-field agglutination has been linked to myelocytic leukemia.22 The RBC membrane change is through somatic mutation resulting in one cell population lacking NeuAc.23

Tn acTivaTion

Tn activation has been described in healthy individuals and some infants. It may be caused by incomplete expression of the gene responsible for converting the Tn into T expressed

C. Melland and C. Hintz


on RBCs.24,25 Incomplete synthesis of T on RBCs in this type of polyagglutination causes agglutination with Salvia sclarea, Salvia horminum, and Glycine soja.26

Indications

Indications for polyagglutination can come from discrepant results obtained during ABO testing caused by the naturally occurring antibody (e.g., anti-T) in human polyclonal antisera.27 Since the advent of monoclonal reagents, however, most examples of polyagglutination are only suspected after reviewing the clinical findings of the patient who has suspected hemolysis or anemia of unknown origin. Lectins and normal adult human sera can be used to detect the surface changes on polyagglutinable RBCs. By observing the patterns of reactivity of the RBCs in question with human sera and with the various lectins, the specific polyagglutinable state of the patient’s RBCs can be identified.

Materials

Prepare human sera for testing against patient RBCs by pooling the plasma or serum from four to six group AB donors drawn within 48 hours. This plasma pool can be stored frozen for later use.26

Pooled serum from two to three cord blood samples can be used as a negative control for the polyagglutination screen. Mix one drop each of group A1 and group B RBCs with two drops of pooled cord sera. Incubate at room temperature for approximately 5 minutes. Centrifuge, and read macroscopically for agglutination. The cord sera should not agglutinate the group A1 or group B RBCs. If there is reactivity, cord sera are contaminated with maternal antibody and should not be used.28

If lectins are not available commercially, they can be extracted from a variety of seeds.29 Salvia horminum lectin is also known as annual sage, Salvia sclarea is clary sage, Arachis hypogea is from raw peanuts, and Glycine soja (also known as Glycine max) is from wild soybean. To prepare the lectins, measure 1 g of seeds. Wash, and grind up the seeds and then incubate in 5 mL of 0.9 percent saline. Let the mixture sit overnight. Centrifuge the mixture, and harvest the supernatant.30 To obtain a clear supernatant, run the lectin through a filter.

Quality Control for In-House Prepared LectinsAlthough it is relatively easy to create lectins, it is difficult

to find polyagglutinable RBCs to use as positive controls

to ensure potency and specificity. T-activated RBCs can be prepared by treating normal RBCs with neuraminidase. These RBCs can then be used as a positive control for Arachis hypogea, for Glycine soja, and for the pooled human plasma.

To prepare T-activated RBCs, wash adult group O RBCs from a single donor source with 0.9 percent saline three to four times. Dilute one part neuraminidase with nine parts phosphate-buffered saline (PBS) (i.e., one drop of neuraminidase with nine drops of PBS). In one tube, mix one volume (e.g., three drops) of the packed RBCs with an equal volume of diluted neuraminidase. In a second tube, mix one volume (e.g., three drops) of the packed RBCs with an equal volume of 6 percent albumin to serve as a negative control when testing to ensure T-activation has occurred. Incubate both tubes at 37°C for approximately 15 minutes, then wash three to four times with 0.9 percent saline, and create a 3–5 percent concentration in 0.9 percent saline.

Test the treated and untreated RBCs with Glycine soja to ensure activation of the treated cells and no activation with the untreated cells. T-activated RBCs can be stored in liquid nitrogen if not used immediately for testing.

Quality Control

To prepare the positive control, add one drop of T-activated RBCs to one drop of each lectin tested. To prepare the negative control, add one drop of cord sera to a 3–5 percent suspension of the patient’s RBCs. Mix, and incubate all tubes using the same time and temperature as specified in the manufacturer’s instructions or as determined in the in-house standardization of the lectin in use. Centrifuge, and read macroscopically for agglutination.

The T-activated RBCs will react with Arachis hypogea, Glycine soja, and the pooled plasma/sera control but will not react with Salvia sclarea, Salvia horminum/Dolichos biflores, or pooled cord sera. See Table 1.27,30

Procedure

Mix one drop of a 3–5 percent suspension of the patient’s RBCs with two drops of pooled donor plasma or sera. If lectins are commercially available, mix one drop of the patient’s RBC suspension with each lectin following the manufacturer’s instructions for amount, time, and temperature indicated. If in-house prepared lectins are used, the amount of lectin would be dependent upon in-house standardization. Centrifuge, and read macroscopically for agglutination.

Detecting polyagglutinable RBCs


C. Melland and C. Hintz

Interpretation

If adult sera are reactive with the RBCs tested and the cord sera are nonreactive, the RBCs are polyagglutinable. If the adult sera are nonreactive and the cord sera are nonreactive, then the RBCs are not polyagglutinable. If the cord sera are reactive, then consider spontaneous agglutination due to antibodies present in the plasma.

Note that additional lectins are available to help determine the specific type of polyagglutination present. Table 1 shows examples of lectin reactivity with examples of polyagglutinable RBCs.15,31

Limitations

Lectins kits are no longer available from commercial manufacturers. Noncommercial lectins should be used for testing with the appropriate controls tested the day of use. Cad and Tn polyagglutinable RBCs are needed to create positive controls for Salvia horminum and Salvia sclarea. The unavailability of these polyagglutinable RBCs is problematic for using these lectins. If lectins are not made with the proper solutes, they cannot be stored frozen.

Patient RBCs that are not completely polyagglutinated or are transiently polyagglutinable may not react by testing with lectins or human sera. Conversely, some lectins have antibacterial activity and may agglutinate RBCs coated with bacterial polysaccharides. This reactivity is not the same reactivity caused by the state of polyagglutination and justifies the use of pooled human sera to support the polyagglutination test.32

References

1. Sharon N, Lis H. History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 2004;14: 53R–62R.

2. Gorakshakar AC, Ghosh K. Use of lectins in immunohematology. Asian J Transfus Sci 2016;10:12–21.

3. Hubener G. Investigation of isoagglutination, with special consideration of apparent deviations from the blood grouping scheme. [In German] Z Immunitatsforsch 1926;45:223–48.

4. Poulsen PE. Experimentally produced “polyagglutinability” (T-transformation of erythrocytes in vivo) in guinea pigs infected with pneumococci. Nature 1954;173:82–3.

5. Crookston KP, Reiner AP, Cooper LJN, Sacher RA, Blajchman MA, Heddle NM. RBC T activation and hemolysis: implications for pediatric transfusion management. Transfusion 2000;40: 801–12.

6. Levine P, Katzin EM. Temporary agglutinability of red blood cells. Proc Soc Exp Biol NY 1938;39:167–9.

7. Ramasethu J, Luban N. T Activation. Br J Haematol 2001;112: 259–63.

8. Moh-Klaren J, Bodivit G, Jugie M, et al. Severe hemolysis after plasma transfusion in a neonate with necrotizing enterocolitis, Clostridium perfringens infection and red blood cell T-polyagglutination. Transfusion 2017;57:2571–7.

9. Gerbal A, Maslet C, Salmon C. Immunological aspects of the acquired-B antigen. Vox Sang 1975;28:398.

10. Judd WJ, McGuire-Mallory D, Anderson KM, et al. Concomitant T- and Tk-activation association with acquired-B antigens. Transfusion 1979;19:293–8.

11. Doinel C, Andreu G, Cartron JP, Salmon C, Fukuda MV. Tk polyagglutination produced by in vitro by an endo–beta–galacto–sidase. Vox Sang 1980;38:94–8.

12. Bird GW, Wingham J, Beck ML, Pierce SR, Oates GD, Pollock A. Th, a “new” form of erythrocyte polyagglutinability. Lancet 1978;1:1215-6.

13. Bird GW, Windham J, Seger R, Kenny AB. Tx, a “new” red cell cryptantigen exposed by pneumococcal enzymes. Blood Transf Immunohaemat 1982;25215–16.

14. Suchanowska A, Kaczmarek R, Duk M, et al. A single point mutation in the gene encoding Gb3/CD77 synthase causes a rare inherited polyagglutination syndrome. J Biol Chem 2012;287:38220–30.

15. Reid ME, Lomas-Francis C, Olsson M. The blood group antigen factsbook. 3rd ed. San Diego, CA: Academic Press, 2012.

16. Halverson GR, Lee AH, Oyen R, Reiss RF, Hurlet-Jensen A, Reid ME. Altered glycosylation leads to Tr polyagglutination. Transfusion 2004;44:1588–92.

17. Mawby WJ, Tanner MJA, Anstee DJ, Clamp JR. Incomplete glycosylation of erythrocyte to membrane protein in congenital dyserythropoietic anemia Type II. (CDA II) Br Haematol 1983;55:357–68.

18. Fukuda MN. HEMPAS: Hereditary erythroblastic multiniclearity with positive acidified serum lysis test. Biochim Biophys Acta 1999;1455:231–9.

19. Sanger R, Gavin J, Tippett P, Teesdale P, Eldon K. Plant agglutinin for another human blood-group. Lancet 1971;i:1130.

Table 1. Examples of lectin reactivity with examples of polyagglutinable RBCs

LectinNormal RBCs

Polyagglutinable RBCs

Cad+ Tn+ T+ Tk+ NOR HEMPAS

Arachis hypogea 0 0 0 + + 0 0

Salvia sclarea 0 0 + 0 0 0 0

Salvia horminum/Dolichos biflores

0 + + 0 0 0 0

Glycine soja 0 + + + 0 0 +

Pooled plasma/sera control

0 + + + + + +

Pooled cord sera 0 0 0 0 0 0 0



Detecting polyagglutinable RBCs

20. Pickles MM, Morton JA. The Sda blood group antigen. In: Human blood groups. 5th International Convocation on Immunology, Buffalo, NY, 1977:277–86.

21. Sringarm S, Chiewsilp P, Tubrod J. Cad receptor in Thai blood donors. Vox Sang 1974;26:462–6.

22. Bird GW, Wingham J, Pippard MJ, Hoult JG, Melikian V. Erythrocyte membrane modification in malignant diseases of myeloid and lymphoreticular tissues. I. Tn–polyagglutination in acute myelocytic leukaemia. Br J Haematol 1976;33: 289–94.

23. Cartron JP, Cartron J, Andreu G, et al. Selective deficiency of 3-B-galactosyl-transferase (T-transferase) in Tn-polyagglutinable erythrocytes. Lancet 1978;i:856–7.

24. Beck ML. Tn: a nonmicrobial form of polyagglutination. In: Beck ML, Judd WJ, Eds. Polyagglutination. Washington, DC: AABB, 1980:55–70.

25. Schulz M, Fortes P, Brewer L, et al. “In Utero” exposure of Tn and Th cryptantigens (abstract). Transfusion 1983;23:422.

26. Judd WJ. Judd’s methods in immunohematology. 3rd ed. Durham, NC: Montgomery Scientific Publications, 2008.

27. Judd WJ. Review: polyagglutination. Immunohematology 1992;8:58–69.

28. Judd, WJ. The use of purified lectins in immunohematology. Transfusion 1979;19:769–9.

29. King MJ, Liew YW, Moores PP, Bird GWG. Enhanced reaction with Vicia graminea lectin and exposed terminal N-acetyl-D-glucosaminyl residues on a sample of human red cells with Hb M-Hyde Park. Transfusion 1988;28:549–55.

30. Mollison PL, Engelfriet CP, Contreras M. Blood transfusion in clinical medicine. 10th ed. Oxford, UK: Blackwell Science, 1997.

31. Issitt PD. Applied blood group serology. 3rd ed. Miami, FL: Montgomery Scientific iPublications, 1985:457.

32. Hemo bioscience. Lectin package insert. Version 2.1 Revised 03-2015. Morrisville, NC: Hemo bioscience.

Cami Melland, MLS(ASCP)SBBCM, Clinical Laboratory Scientist, Manager (corresponding author), Immunohematology Reference Laboratory, Bonfils Blood Center, 717 Yosemite Street, Denver, CO 80230, [email protected]; and Connie Hintz, MT(ASCP)SBB, Clinical Laboratory Scientist, Immunohematology Reference Laboratory, Bonfils Blood Center, Denver, CO.

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The editorial staff of Immunohematology welcomes manuscripts pertaining to blood group serology and molecular genetics for consideration for publication. We are especially interested in review articles, case reports, papers on platelet and white cell serology, scientific articles covering original investigations or new blood group alleles, papers on molecular testing, and papers on new methods for use in the blood bank. To obtain instructions for

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Marjory Stroup Walters1924–2018

Marjory was born in 1924 in Iola, Kansas. She was the sixth D+ child of a D– mother, but fortunately was unaffected by Rh antibodies. She received her medical technology degree in 1947 and worked as a medical technologist at the University of Kansas Hospital.

Marjory joined Ortho Pharmaceutical Corporation (Raritan, NJ) in 1951, working first in Quality Assurance and manufacturing blood bank antisera before moving to the Blood Consultation Service. The Blood Consultation Service/ Philip Levine Laboratory had been established after the discovery of Rh and its role in erythroblastosis fetalis. The Blood Consultation Service rapidly became a world-recognized center for immunohematology research, investigating approximately 12,000 immunohematology problems per year, as well as becoming a center for immunohematology education.

A generation of blood bank technologists learned immunohematology at the Ortho Immunohematology Schools under Marjory’s leadership. She was a mentor and friend to many of the leading lights in blood banking in the late 20th and early 21st centuries and inspired many technologists to become “blood bankers.”

Marjory was the author and co-author with Margaret Treacy of numerous publications including the Ortho “Red Books” on The ABO & Rh Systems, Compatibility Testing, and Hemolytic Disease of the Newborn. In 1982, Blood Group Antigens and Antibodies by Marjory and Margaret continued immunohematology education. She was a sought-after lecturer and educator and traveled the world speaking about blood banking topics and conducting workshops. “Have knit dress, will travel,” said Marjory. Marjory and other leading immunohematologists found that they were always the ones giving presentations but never had the opportunity to learn much themselves, so Marjory and Shirley Busch decided that a special meeting of reference-lab level immunohematologists was needed. Thus was born the first sessions of the Reference Lab meetings, sponsored by the AABB, which later separated from AABB and became the Invitational Conference of Investigative Immunohematologists (ICII). It continues today as a leading gathering of blood bank specialists.

Marjory’s red blood cells (RBCs) were positive for many important antigens, so they were used in the laboratory as a screening cell. At first it had been thought that Marjory was D–, even when she had donated blood. But further attention showed she was D+, having a D suppressed by her C. As screening cells, Marjory’s RBCs were useful, especially being Le(a+) and very strongly P1+. Every now and then, however, her RBCs would be reactive when no other cells were. Samples were sent to Robert Race and Ruth Sanger in London, who found them also to be Cw+.

During her 30+ years in blood banking, Marjory made innumerable discoveries and important observations. She showed that Jsa was part of the Kell system. She described the first examples of anti-Cra, -Jra, -Lu8, -Lu13, -K12, -K13, and -K19. She was involved in extensive work on several systems including Diego, Kell, and Lutheran.

in MeMORiaM


Marjory was an active member of AABB for more than 50 years. Among her numerous awards were two AABB honors: the Ivor Dunsford award in 1973 and the Emily Cooley award in 1983.

Marjory was a pioneer in the field, and her work contributed to the modern practice of transfusion medicine. She will be remembered by scientists, business colleagues, and friends, not only for her significant contributions, but also for her commitment to quality, leadership, and education delivered with an irrepressible spirit and enthusiasm.

AcknowledgmentsThe authors thank Steve Pierce whose extensive knowledge and work on the history of blood banking contributed to this piece.

About the AuthorsJoAnn Hegarty, MT(ASCP)SBB, Ortho Clinical Diagnostics (Formerly); and Tony S. Casina, MT(ASCP)SBB, (corresponding

author), Ortho Clinical Diagnostics, 1001 U.S. Highway 202 South, Raritan, NJ 08869, [email protected].

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annOunCeMents

The Johns Hopkins Hospital Specialist in Blood Bank Technology Program

The Johns Hopkins Hospital was founded in 1889. It is located in Baltimore, MD, on the original founding site, just 45 minutes from Washington, DC. There are approximately 1,000 inpatient beds and another 1,200 outpatient visits daily; nearly 600,000 patients are treated each year.

The Johns Hopkins Hospital Transfusion Medicine Division is one of the busiest in the country and can provide opportunities to perform tasks that represent the entire spectrum of Immunohematology and Transfusion Medicine practice. It provides comprehensive support to all routine and specialized areas of care for surgery, oncology, cardiac, obstetrics, neonatal and pediatric, solid organ and bone marrow transplant, therapeutic apheresis, and patients with hematological disorders to name a few. Our intradepartment Immunohematology Reference Laboratory provides resolution of complex serologic problems, transfusion management, platelet antibody, and molecular genotype testing.

The Johns Hopkins Hospital Specialist in Blood Bank Technology Program is an onsite work-study, graduate-level training program for certified Medical Technologists, Medical Laboratory Scientists, and Technologists in Blood Banking with at least 2 years of full-time Blood Bank experience.

The variety of patients, the size, and the general intellectual environment of the hospital provide excellent opportunities for training in Blood Banking. It is a challenging program that will prepare competent and knowledgeable graduates who will be able to effectively apply practical and theoretical skills in a variety of employment settings. The Johns Hopkins Hospital Specialist in Blood Bank Technology Program is accredited by the Commission on Accreditation of Allied Health Education Programs (CAAHEP). Please visit our Web site at http://pathology.jhu.edu/department/divisions/transfusion/sbb.cfm for additional information.

Contact: Lorraine N. Blagg, MA, MLS(ASCP)CMSBB Program Director E-mail: [email protected] Phone: (410) 502-9584

The Johns Hopkins Hospital Department of Pathology Division of Transfusion Medicine Sheikh Zayed Tower, Room 3100 1800 Orleans Street Baltimore, MD 21287

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Masters of Science (MSc) in Transfusion and Transplantation Sciences at the University of Bristol, England

Applications are invited from medical or science graduates for the Master of Science (MSc) degree in Transfusion and Transplantation Sciences at the University of Bristol. The course starts in October 2018 and will last for 1 year. A part-time option lasting 2 or 3 years is also available. There may also be opportunities to continue studies for PhD or MD following the MSc. The syllabus is organized jointly by the Bristol Institute for Transfusion Sciences and the University of Bristol, Department of Pathology and Microbiology. It includes:

• Scientific principles of transfusion and transplantation

• Clinical applications of these principles

• Practical techniques in transfusion and transplantation

• Principles of study design and biostatistics

• An original research project

Application can also be made for a Diploma in Transfusion and Transplantation Sciences or a Certificate in Transfusion and Transplantation Sciences.

The course is accredited by the Institute of Biomedical Sciences.

Further information can be obtained from the Web site:http://ibgrl.blood.co.uk/MSc/MscHome.htm

For further details and application forms, please contact:

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Online Specialist in Blood Bank (SBB)Certificate and Masters in Clinical LaboratoryManagement ProgramRush University | College of Health SciencesContinue to work and earn graduate credit while the Rush University SBB/MS program prepares you for the SBB exam and the Diplomat in Laboratory Management (DLM) exam given by ASCP Board of Certification! (Please note acceptable clinical experience is required for these exams.)

Rush University offers online graduate level courses to help you achieve your career goals.

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